Apparatus and methods for the automated synthesis of small molecules

ABSTRACT

Provided are methods for purifying N-methyliminodiacetic acid (MIDA) boronates from solution. Also provided are methods for deprotection of boronic acids from their MIDA ligands. The purification and deprotection methods can be used in conjunction with methods for coupling or otherwise reacting boronic acids. Iterative cycles of deprotection, coupling, and purification can be performed to synthesize chemical compounds of interest. The methods are suitable for use in an automated chemical synthesis process. Also provided is an automated small molecule synthesizer apparatus for performing automated synthesis of small molecules using iterative cycles of deprotection, coupling, and purification in accordance with methods of the invention. Coupling and other reactions embraced by the invention include, without limitation, Suzuki-Miyaura coupling, oxidation, Swern oxidation, “Jones reagents” oxidation, reduction, Evans&#39; aldol reaction, HWE olefination, Takai olefination, alcohol silylation, desilylation, p-methoxybenzylation, iodination, Negishi cross-coupling, Heck coupling, Miyaura borylation, Stille coupling, and Sonogashira coupling.

RELATED APPLICATIONS

This application is a continuation of U.S. 371 application Ser. No.13/811,527, filed Mar. 14, 2013, now U.S. Pat. No. 9,238,597; which isthe U.S. national phase of International Patent Application No.PCT/US2011/045064, filed Jul. 22, 2011; which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/367,176,filed Jul. 23, 2010.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under NationalInstitutes of Health Grant Nos. GM080436 and GM090153. The governmenthas certain rights in the invention.

BACKGROUND

Similar to peptides, oligonucleotides, and oligosaccharides, most smallmolecule natural products are highly modular in their constitution. Thisis because, like the aforementioned oligomers, the majority of smallmolecules are biosynthesized via the sequential coupling of bifunctionalbuilding blocks. Specifically, polyketides are derived from multiplemalonyl-CoA and/or methmalonyl-CoA units, non-ribosomal peptides arebuilt from amino acids, polyterpenes are stitched together fromisopentenyl pyrophosphate and/or dimethylallyl pyrophosphate buildingblocks, and fatty acids are prepared from fragments of malonyl-CoA.Other classes of modular natural products result from the oxidativecoupling of common building blocks, such as shikimic acid, amino acids,and/or their respective derivatives.

With peptides, oligonucleotides, and increasingly oligosaccharides, thisinherent modularity is now routinely harnessed to enable fully automatedsyntheses from suitably protected bifunctional building blocks (R. B.Merrifield, Science 1965, 150, 178-185; M. H. Caruthers, Science 1985,24, 799; and O. J. Plante, M. R. Palmacci, P. H. Seeberger, Science2001, 291, 1523). As a direct result of these advances, research intheses areas is primarily focused on discovering and understanding newmolecular function. In stark contrast, despite tremendous advances overthe course of nearly two centuries, the laboratory synthesis of smallmolecules remains a relatively complex, inflexible, and non-systematizedprocess practiced almost exclusively by highly-trained specialists. (Forpioneering developments in the automated synthesis of small moleculesvia polymer-assistance and/or flow chemistry, see: a) C. H. Hornung, M.R. Mackley, I. R. Baxendale and S. V. Ley and, Org. Proc. Res. Dev.2007, 11, 399-405; b) Nikzad Nikbin, Mark Ladlow, and Steven V. Ley Org.Process Res. Dev. 2007, 11, 458-462; and c) France, S.; Bernstein, D.;Weatherwax, A.; Lectka, T. Org. Lett. 2005, 7, 3009-3012.) Thus,research in this area is still heavily weighted towards synthesis. Giventhe special properties of many small molecules that make them uniquelysuited for a wide range of applications in science, engineering, andmedicine, increased access to these compounds via a highly general andautomated synthesis platform that is accessible to the non-expert wouldbe highly enabling. Ultimately, such a process could help shift theprimary focus from the synthesis of small molecules to the discovery andunderstanding of important small molecule functions.

SUMMARY

Certain aspects of the invention relate to an apparatus that utilizes acarbon-carbon bond-forming reaction iteratively to assemble a wide rangeof small molecules from pre-fabricated building blocks, and methods forusing the same. In certain embodiments, analogous to the automatedpreparation of peptides from suitably protected amino acids, theautomated process involves the controlled, iterative assembly ofbifunctional haloboronic acid building blocks protected as thecorresponding N-methyliminodiacetic acid (MIDA) boronates. In certainembodiments, obviating the need for any covalent attachment to a solidsupport, purification of intermediates is achieved by harnessing tworemarkably general physical properties of MIDA boronates: the capacityfor catch-and-release chromatography with silica gel; and theirinsolubility in hexanes. Additional aspects, embodiments, and advantagesof the invention are discussed below in detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts analogous strategies for the synthesis of peptides andsmall molecules.

FIG. 1B is a schematic representation of exemplary iterated cycles ofdeprotection, cross-coupling, and purification with a finaldirect-release cross-coupling step, wherein for each coupling, roughly 3equivalents of boronic acid are employed relative to each halide.

FIG. 1C depicts two distinct strategies for purifying MIDA boronateintermediates. The first strategy is a “catch-and-release purification”and takes advantage of the high affinity of MIDA boronates for silicagel. Specifically, a crude product mixture is loaded onto a pad ofsilica gel, which is then flushed with Et₂O/MeOH. While all byproductsare rapidly eluted in this polar solvent mixture, MIDA boronates showessentially infinite retention. Remarkably, the “catch” phenomenon isgeneral for any compound that contains the MIDA boronate functionalgroup. Simply switching the solvent to tetrahydrofuran (THF), however,“releases” the purified MIDA boronate as a solution suitable for use inthe subsequent deprotection reaction. The second strategy is a“precipitation purification” and harnesses the general insolubility ofMIDA boronates in hexanes. Specifically, a crude reaction mixture in THFis transferred into a chamber containing hexanes. The MIDA boronateprecipitates, and is separated from the soluble reaction byproducts viafiltration. A customized hybrid purification vessel may be used toharness both of these purification processes in series, thus providing ahighly robust and general method for automated purification of MIDAboronate intermediates without the need for covalent attachment to asolid support.

FIG. 1D depicts a photograph of one embodiment of a fully automatedsmall molecule synthesizer comprising modules for (i) deprotection, (ii)cross-coupling, and (iii) purification, all of which are under thecontrol of a computer equipped with custom-designed software.

FIG. 2 depicts examples of reactions which may be run in a synthesizer,wherein D represents a deprotection step, CC represents a cross-couplingstep, P represents a purification step, and RCC represents across-coupling step with either fast or slow in situ release of theboronic acid/boronic ester.

FIG. 3 depicts selected building blocks, including bifunctionalhaloboronic acid building blocks protected as the correspondingN-methyliminodiacetic acid (MIDA) boronates, and compounds which can beprepared from said building blocks.

FIG. 4 depicts selected building blocks, including bifunctionalhaloboronic acid building blocks protected as the correspondingN-methyliminodiacetic acid (MIDA) boronates, and compounds which can beprepared from said building blocks.

FIG. 5 depicts (top) a design schematic of one embodiment of anautomated small molecule synthesizer and (bottom) an example of theconnectivity of the various pumps, valves, ports and tubes; wherein (1)denotes solvent reservoirs; (2) denotes a drying and degassing table;(3) denotes a heating block and stir-plate; (4) denotes solenoid valvesand gas manifolds; (5) denotes a deprotection table; (6) denotes apurification table; (7) denotes a valve module (with one example of avalve map shown in FIG. 7); (8) denotes main syringe pumps; (9) denotesa syringe pump for purification; and (10) denotes a syringe pump foraqueous reactions.

FIG. 6 depicts photographs of exemplary reaction tubes, tubing andfittings.

FIG. 7 depicts an example of a valve map.

FIG. 8 depicts an example of a reaction tube.

FIG. 9 is a schematic of an example of an aqueous deprotection module.

FIG. 10 depicts an example of a precipitation chamber and silica column.

FIG. 11 depicts an example of a drying and degassing tube.

FIG. 12 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 13 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 14 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 15 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 16 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 17 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 18 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 19 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 20 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 21 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 22 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 23 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 24 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 25 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 26 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 27 is a pair of ¹H NMR spectra corresponding to (i) a mock reactionmixture comprising a MIDA boronate, and (ii) the MIDA boronate afterpurification from the mixture.

FIG. 28 depicts (A) automated aqueous deprotection of phenyl MIDAboronate and subsequent automated cross-coupling of phenyl boronic acidwith a vinyl iodide bifunctional building block; (B), automated aqueousdeprotection of trienyl MIDA boronate and subsequent automatedcross-coupling of trienyl boronic acid with a vinyl iodide bifunctionalbuilding block; and (C) automated aqueous deprotection of butenyl MIDAboronate and subsequent automated cross-coupling of butenyl boronic acidwith an isomeric mixture of dienyl vinyl iodide bifunctional buildingblocks.

FIG. 29 depicts fully automated synthesis of all-trans-retinal using anaqueous deprotection module.

DETAILED DESCRIPTION

Certain aspects of the present invention are directed to apparatuses andmethods for the automated synthesis of small molecules. In certainembodiments, the small molecules are prepared by using a single reactioniteratively to unite a collection of bifunctional building blocks havingall of the required functionality, oxidation states, and stereochemistrypre-installed.

Bifunctional MIDA-Protected Haloboronic Acids

A key to the development of apparatuses and methods for the automatedsynthesis of small molecules was the use of the Suzuki-Miyaura reactionto achieve the iterative cross-coupling (ICC) of bifunctional“haloboronic acids” (FIG. 1A). However, in order to have an efficientautomatable procedure, the development of a mild and selective methodfor reversibly-attenuating one end of each haloboronic acid was requiredto avoid random oligomerization. In this vein, the apparatuses andmethods described herein take advantage of the finding that thetrivalent ligand N-methyliminodiacetic acid (MIDA) can act as a switchto turn the reactivity of a boronic acid “off” and “on” under very mildconditions (E. P. Gillis, M. D. Burke J. Am. Chem. Soc. 2007, 129,6716-6717; and U.S. Patent Application Publication No. 2009/0030238,which is hereby incorporated by reference in its entirety). Thisproperty of MIDA boronates has made it possible to prepare a variety ofnatural products via repeated cycles involving MIDA boronatedeprotection, selective cross-coupling, and purification (FIG. 1B; S. J.Lee, K. C. Gray, J. S. Paek, M. D. Burke J. Am. Chem. Soc. 2008, 130,466-468; E. P. Gillis, M. D. Burke, J. Am. Chem. Soc. 2008, 130,14084-14085; and E. M. Woerly, A. H. Cherney, E. K. Davis, M. D. Burke,J. Am. Chem. Soc. 2010, 132, 6941-6943). Further enabling their generalutility as building blocks, MIDA boronates are uniformly air-stable,highly crystalline, monomeric, free-flowing solids that are fullycompatible with a wide range of common synthetic reagents and silica gelchromatography (E. P. Gillis, M. D. Burke Aldrichimica Acta 2009, 42,17-27). In addition, more recent advances in methods for preparing MIDAboronate building blocks, and the discovery of their capacity for“slow-release” cross-coupling, have substantially expanded the utilityof the synthesis platforms described herein (G. R. Dick, D. M. Knapp, E.P. Gillis, Org. Lett. 2010, 12, 2314-2317; D. M. Knapp, E. P. Gillis J.Am. Chem. Soc. 2009, 131, 6961-6963; and U.S. Patent ApplicationPublication No. 2010/0121062, which is hereby incorporated by referencein its entirety). In fact, a rapidly growing collection of MIDAboronates, representing many of the substructures that commonly appearin natural products and pharmaceuticals, is now commercially-available.The expanding scope of the Suzuki-Miyaura reaction, which increasinglyincludes Csp³-Csp³ type couplings (M. R. Netherton, G. C. Fu, Adv. Syn.Cat. 2004, 346, 1525-1532), indicates that the potential generality ofthis ICC strategy is substantial.

Purification of MIDA-Protected Organoboronic Acids

Transforming an ICC approach into a fully automated process requires ageneral strategy for purifying the synthetic intermediates. In the caseof peptides, oligonucleotides, and oligosaccharides this problem hasbeen solved by linking the growing oligomer to a solid-support. At theend of each coupling reaction, the desired product is separated fromresidual solvents, reagents, and byproducts via a simple filtration.Albeit highly effective in these contexts, there are two majorlimitations of this purification approach as a foundation for ICC-basedsmall molecule synthesis.

First, this strategy requires a ubiquitous chemical handle that enablescovalent linking of the growing oligomer to the solid-phase. In the caseof peptides, oligonucleotides, and oligosaccharides, the respectivemonomers all conveniently contain a common heteroatom linking element asan inherent component of the targeted structure. In contrast, althoughsome excellent solid-phase linking systems have been developed, smallmolecules are quite structurally diverse, and many lack a commonfunctional group available for attachment to a solid-phase.

Second, selectively coupling boronic acids in the presence of MIDAboronates requires that relatively anhydrous conditions be utilizedbecause MIDA boronates are stable and unreactive under anhydrous basicconditions, but are readily hydrolyzed to yield reactive boronic acidswhen treated with aqueous base. In preliminary studies, it was foundthat translating the chemistry of anhydrous Suzuki-Miyauracross-couplings to the solid-phase can be problematic.

Surprisingly, the inventors have discovered two remarkable physicalproperties of MIDA boronates, allowing the circumvention of both of theaforementioned challenges. Collectively, the two properties have enableda highly effective alternative purification strategy and, thus, allowedthe complete automation of ICC with solution-phase chemistry. The twopurification strategies—“precipitation” and “catch-and-release”—arediscussed in detail below. The two purification strategies can be usedalone or in combination, in which case they may be performedsequentially in either order.

Purification by Precipitation.

One aspect of the invention relates to the discovery that virtually allmolecules containing a MIDA-protected organoboronic acid functionalgroup are insoluble in hexanes:THF (3:1 v/v), while almost all boronicacids, other boronic esters, or related surrogates are soluble in thissolvent system (FIG. 1C). This general physical property of MIDAboronates enables a highly efficient precipitation-based purification.(For background on precipitation-based purification see: H. Perrier, M.Labelle, J. Org. Chem. 1999, 64, 2110-2113; T. Bosanac, C. S. Wilcox,Org. Lett. 2004, 6, 2321-2324; and J. C. Poupon, A. A. Boezio, A. B.Charette, Angew. Chem. Int. Ed. 2006, 45, 1415-1420). Further, becausemost catalyst species and organic halides are soluble in hexanes:THF(3:1), MIDA boronates can be directly purified from cross-couplingreactions, such as anhydrous Suzuki cross-coupling reactions. Merelytransferring a crude product mixture in THF (e.g., from a cross-couplingreaction) to a stirred vessel containing an amount of hexanes which isapproximately three times the total volume of THF to be added results inrapid and quantitative precipitation of the MIDA boronate product whilethe residual unreacted boronic acid, as well as most byproducts andother reaction components, such as palladium and phosphine ligands, allremain soluble in the hexane:THF (3:1 v/v) mixture. Simple filtration ofthis mixture, followed by dissolution of the precipitated MIDA boronatewith THF yields, a solution of semi-purified MIDA boronate.

One aspect of the invention relates to a method of purifying a MIDAboronate from a solution, comprising the steps of diluting with hexanethe solution comprising the MIDA boronate, thereby selectivelyprecipitating the MIDA boronate; and isolating the precipitated MIDAboronate. The hexane can be any isomer of hexane or a mixture ofhexanes. Exemplary isomers of hexane useful in the invention includeunbranched hexane (n-hexane), branched hexanes (e.g., isohexane), andcyclohexane.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the precipitated MIDA boronate isisolated by filtration.

In certain embodiments, the present invention relates to any one of theaforementioned methods, further comprising the step of dissolving theprecipitated MIDA boronate in a polar solvent. In certain embodiments,the present invention relates to any one of the aforementioned methods,further comprising the step of dissolving the precipitated MIDA boronatein THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solution comprising the MIDAboronate is a THF solution.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solution comprising the MIDAboronate is added dropwise to the hexane.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the volume of hexane is between abouttwo and about four times the volume of the solution comprising the MIDAboronate.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the volume of hexane is about threetimes the volume of the solution comprising the MIDA boronate.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solution comprising the MIDAboronate is a crude product mixture from a chemical reaction.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction is selected fromthe group consisting of a Suzuki-Miyaura coupling, an oxidation, a Swernoxidation, a “Jones reagents” oxidation, a reduction, an Evans' aldolreaction, an HWE olefination, a Takai olefination, an alcoholsilylation, a desilylation, a p-methoxybenzylation, an iodination, aNegishi cross-coupling, a Heck coupling, a Miyaura borylation, a Stillecoupling, and a Sonogashira coupling.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction comprises the stepof contacting a MIDA boronate with a reagent, wherein the MIDA boronatecomprises a boron having an sp³ hybridization, a MIDA protecting groupbonded to the boron, and an organic group bonded to the boron through aboron-carbon bond; the organic group is chemically transformed, and theboron is not chemically transformed.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the MIDA boronate is represented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of a hydrogen group and anorganic group.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein R²¹, R²², R²³ and R²⁴ are hydrogen.

Catch-and-Release Purification.

It has further been found that all molecules which contain a MIDAboronate functional group have exceptionally high affinity for silicagel (FIG. 1C). For example, it has been discovered that MIDA boronates,regardless of the nature of the organic group appended to boron, have anR_(f) of essentially zero in hexanes:THF (3:1 v/v), Et₂O, and Et₂O:MeOH(98.5:1.5 v/v). Therefore, MIDA boronates can be used as a universal tagfor catch-and-release purification on silica gel. (For an excellentreview on tagging strategies for separations in organic synthesis, see:J. Yoshida, K. Itami, Chem. Rev. 2002, 102, 3693-3716. For an excellentreview on modern separation techniques in organic synthesis, see: C. C.Tzschucke, C. Markert, W. Bannwarth, S. Roller, A. Hebel, R. Haag,Angew. Chem. Int. Ed. 2002, 41, 3964-4000. See, also, D. P. Curran,Angew. Chem. Int. Ed. 1998, 37, 1174-1196; P. H. Toy, K. D. Janda, Acc.Chem. Res. 2000, 33, 546-554; S. V. Ley, A. Massi, F. Rodriguez, D. C.Horwell, R. A. Lewthwaite, M. C. Pritchard, A. M. Reid, Angew. Chem.Int. Ed. 2001, 40, 1053-1055; A. R. Brown, S. L. Irving, R. Ramage, G.Raphy Tetrahedron 1995, 51, 11815-11830; L. A. Thompson, Curr. Opin.Chem. Bio. 2000, 4, 324-337; and M. G. Siegel, P. J. Hahn, B. A.Dressman, J. E. Fritz, J. R. Grunwell, S. W. Kaldor, Tetrahedron Lett.1997, 38, 3357-3360. For the use of catch-and-release type methods topurify proteins, see: J. Porath, J. Carlsson, I. Olsson, G. Belfrage,Nature 1975, 278, 598.) In other words, the MIDA boronate functionalgroup, which is conveniently present in all intermediates utilized incertain ICC sequences (FIG. 1B), enables the reversible non-covalentattachment of any MIDA boronate to silica gel, a solid support.

The use of hexanes:THF (e.g., 3:1 v/v) as a solvent system is importantsince it provides a means (via diluting with hexanes) to purify directlyTHF reaction solutions containing MIDA boronates. With regards toautomated synthesis, as discussed below, this feature is importantbecause advanced manipulations, such as solvent evaporation, are notrequired to prepare the reaction solution for purification. The use ofEt₂O is important because in certain coupling reactions almost everyother compound present in the reaction solution elutes in Et₂O.Interestingly, the addition of 1.5% MeOH (v/v) to the Et₂O ensures thateven polar boronic acids are eluted off of the column with a reasonableamount of solvent. The compatibility of MeOH with MIDA boronates in thepurification method was unexpected since MeOH can be used to deprotectMIDA boronates to the corresponding boronic acid at room temperature.All of the above-mentioned properties have been tested with many MIDAboronates and have been shown to be general. For example, boronates Aand B behave as described above, despite the fact that they arenon-polar and elute well in other solvent combinations, such ashexanes:EtOAc.

Once the unreacted boronic acids, as well as reaction regents, have beeneluted, pure MIDA boronates generally elute well in THF. Also, MIDAboronates generally elute well with MeCN and acetone.

One aspect of the invention relates to a method of purifying a MIDAboronate from a solution, comprising the steps of passing the solutionthrough a silica plug; passing a first liquid through the silica plug;and passing a second liquid through the silica plug, thereby eluting theMIDA boronate in the second liquid; wherein the first liquid containsdiethyl ether or the polarity of the first liquid is less than or equalto about the polarity of a mixture of 98.5:1.5 (v/v) Et₂O:MeOH; and thepolarity of the second liquid is greater than or equal to about thepolarity of THF.

MIDA boronates, like most organic compounds, generally elute morerapidly off of SiO₂ (i.e., have a higher R_(f)) when the polarity of thesolvent is higher. However, the purification method described abovetakes advantage of special properties of MIDA boronates in certainsolvents. Specifically, there are certain solvent systems in which theR_(f) of a MIDA boronate is not related to the polarity of the solvent.In fact, in certain solvent systems the R_(f) can approach or be zero.For example, even though chloroform is more polar than THF, the R_(f) ofdodecyl MIDA boronate in chloroform is 0.00 and in THF is 0.80. Whilenot intending to be bound by any particular theory, this very surprisingphenomenon likely involves a unique interaction between all threefactors: the solvent, silica gel and MIDA boronate. Thus, it is possibleto isolate a MIDA boronate on a silica column if one picks a solventthat is an exception to the elution rules (such as chloroform or Et₂O).To remove a MIDA boronate from the column thus loaded, one switches to apolar solvent that obeys the normal elution rules (such as THF, MeCN, oracetone).

It has also been found that a functionalized silica gel, such as3-aminopropyl-functionalized silica gel, can be substituted for SiO₂without affecting the properties of the MIDA boronate/SiO₂ interaction.The functionalized silica gel can be used to scavenge, for example,metal catalysts from the solution. Therefore, in certain embodiments,the present invention relates to any one of the aforementioned methods,wherein the silica is 3-aminopropyl-functionalized silica.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid comprises diethylether.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid is diethyl ether.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid is a mixture of diethylether and methanol.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein-first liquid is a mixture of diethylether and methanol; and the ratio of diethyl ether to methanol is98.5:1.5 (v/v).

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF, MeCN, ethylacetate or acetone, or a solvent of similar polarity.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF, MeCN, ethylacetate or acetone.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solution is a crude product mixturefrom a chemical reaction.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction is selected fromthe group consisting of a Suzuki-Miyaura coupling, an oxidation, a Swernoxidation, a “Jones reagents” oxidation, a reduction, an Evans' aldolreaction, an HWE olefination, a Takai olefination, an alcoholsilylation, a desilylation, a p-methoxybenzylation, an iodination, aNegishi cross-coupling, a Heck coupling, a Miyaura borylation, a Stillecoupling, and a Sonogashira coupling.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction comprises thesteps of contacting a MIDA boronate with a reagent, wherein the MIDAboronate comprises a boron having an sp³ hybridization, a MIDAprotecting group bonded to the boron, and an organic group bonded to theboron through a boron-carbon bond; the organic group is chemicallytransformed, and the boron is not chemically transformed.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the MIDA boronate is represented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of a hydrogen group and anorganic group.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein R²¹, R²², R₂₃ and R²⁴ are hydrogen.

Combination Precipitation & Catch-and-Release Purification.

The two purification strategies discussed above can be combined into onerobust and general process. Specifically, the solution which issubjected to the catch-and-release purification described above can be asolution which is derived from the selective precipitation of a MIDAboronate.

One aspect of the invention relates to a method of purifying a MIDAboronate from a solution, comprising the steps of diluting the solutionwith hexane, thereby selectively precipitating the MIDA boronate;passing the diluted solution through a silica plug, thereby depositingthe precipitated MIDA-protected organoboronic acid on the silica plug;passing a first liquid through the silica plug; and passing a secondliquid through the silica plug, thereby eluting the MIDA boronate in thesecond liquid; wherein the first liquid contains diethyl ether or thepolarity of the first liquid is less than or equal to about the polarityof a mixture of 98.5:1.5 (v/v) Et₂O:MeOH; and the polarity of the secondliquid is greater than or equal to about the polarity of THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid comprises diethylether.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid is diethyl ether.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the first liquid is a mixture of diethylether and methanol.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein first liquid is a mixture of diethylether and methanol; and the ratio of diethyl ether to methanol is98.5:1.5 (v/v).

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF, MeCN, ethylacetate or acetone, or a solvent of similar or greater polarity.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF, MeCN, ethylacetate or acetone.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the second liquid is THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solution is a crude product mixturefrom a chemical reaction.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction is selected fromthe group consisting of a Suzuki-Miyaura coupling, an oxidation, a Swernoxidation, a “Jones reagents” oxidation, a reduction, an Evans' aldolreaction, an HWE olefination, a Takai olefination, an alcoholsilylation, a desilylation, a p-methoxybenzylation, an iodination, aNegishi cross-coupling, a Heck coupling, a Miyaura borylation, a Stillecoupling, and a Sonogashira coupling.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the chemical reaction comprises thesteps of contacting a MIDA boronate with a reagent, wherein the MIDAboronate comprises a boron having an sp³ hybridization, a MIDAprotecting group bonded to the boron, and an organic group bonded to theboron through a boron-carbon bond; the organic group is chemicallytransformed, and the boron is not chemically transformed.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the MIDA boronate is represented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of a hydrogen group and anorganic group.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein R²¹, R²², R²³ and R²⁴ are hydrogen.

Customized Hybrid Purification Vessels.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein a customized hybrid purification vesselwhich contains both a “precipitation chamber” and a “catch-and-releasechamber” arranged in series (FIG. 1C) is used. In this system, a crudecross-coupling reaction is transferred to a first (e.g., upper) chamberfilled with hexanes, resulting in rapid and quantitative precipitationof the MIDA boronate-containing product while the residual boronic acid(and most byproducts), palladium, and phosphine ligand all remainsoluble. As previously noted, simple filtration of this suspension,followed by washing with Et₂O:MeOH, places the resulting semi-purified,solid MIDA boronate on top of a silica gel plug that resides in a second(e.g., lower) chamber. This lower chamber is then subjected to washingwith copious volumes of, for example, Et₂O:MeOH 98.5:1.5 (v/v) followedby a defined small volume of THF to effect the catch-and-release silicagel purification. The resulting THF solution of purified MIDA boronateis conveniently ready for utilization in subsequent cycles ofdeprotection and coupling.

Purification/Deprotection of MIDA-Protected Organoboronic Acids

The challenges associated with purifying boronic acids include the factthat “the polar and often amphiphilic character tends to make theirisolation and purification difficult” (Hall, D. G. Boronic Acids;Wiley-VCH: Weinheim, Germany, 2005; pp 57-58). Further, “[t]he widelyknown and used boronic acids show variable stability (vinyl-, alkyl-,and alkynylboronic acid are not very stable), and their purification isnot straightforward. Moreover, isolated boronic acids generally containlarge quantities of anhydrides or boroxines, which result in problemsfor determining their stoichiometry” (Darses, S.; Genet, J-P. Chem. Rev.2008, 108, 288-325).

A number of approaches for purifying boronic acids have been developed,but all are limited in their generality. The most basic approach is torecrystallize the boronic acid, typically from an aqueous solution.However, this approach is only efficient if the sample is alreadyrelatively pure and when the temperature-dependent solubility of theboronic acid in water is favorable. When non-polar recrystallizationsolvents are employed, significant dehydration of the boronic acid toafford the boroxine can occur. (Santucci, L.; Gilman, H. J. Am. Chem.Soc. 1958, 80, 193-196). Another approach is “phase switching”liquid/liquid partitioning (Mothana, S.; Grassot, J-M.; Hall, D. G.Angew. Chem. Int. Ed. 2010, 49, 2883-2887). In this approach the boronicacid is converted into the anionic borate species in strong base (pH10), non-anionic organics are washed away, and then the solution isacidified (pH 1-5) to regenerate the boronic acid. This method is notcompatible with boronic acids containing acidic functional groups, basicfunctional groups, or any functionality that is acid- or base-sensitive,including the boronic acid functionality. A solid supported scavengerfor boronic acids based on diethanolamine, abbreviated DEAM-PS, has alsobeen reported (Hall, D. G.; Tailor, J.; Gravel, M. Angew. Chem. Int. Ed.1999, 38, 3064-3067). However, this method is expensive and does notrepresent a practical or scalable solution.

Boronic acids can be purified in a two-step process with theintermediacy of a boronic acid surrogate. For example, boronic acids canbe converted to the corresponding trifluoroborate salt which can becrystallized (Darses, S.; Genet, J-P. Chem. Rev. 2008, 108, 288-325).However, limitations of this approach include the fact that thecrystallization conditions are substrate-specific, large amounts offluoride are used, some impurities co-crystallize with the product, andregenerating the boronic acid from the trifluoroborate is not efficient(Molander, G. A.; Cavalcanti, L. N.; Canturk, B.; Pan, P-S.; Kennedy, L.E. J. Org. Chem. 2009, 74, 7364-7369). Alternatively, boronic acids canbe dehydrated in the presence of a diol (most often pinacol) to form thecorresponding boronic ester. Some aryl boronic esters have morefavorable chromatography, extraction, and crystallization propertiesthan the corresponding boronic acids. However other classes of boronicesters (heteroaryl, alkenyl, alkyl, alkynyl, etc.) tend to have highlyvariable features. Further, as the boronic ester becomes stable enoughto improve its purification properties, the conditions required toregenerate the boronic acid become harsher. For example, converting apinacol boronic ester to the corresponding boronic acid typicallyrequires aqueous acid and an oxidant (often NaIO₄), which limits thegenerality of this approach (Murphy, J. M.; Tzschuck, C. C.; Hartwig, J.F. Org. Lett. 2007, 9, 757-760).

Finally, unstable boronic acids present a particularly challengingproblem. None of the above-mentioned approaches can be used to purifyunstable boronic acids, such as vinyl boronic acids. Remarkably, vinylboronic acid can be generated from vinyl MIDA boronate in greater than95% purity (Knapp, D. M.; Gillis, E. P.; Burke, M. D. J. Am. Chem. Soc.2009, 131, 6961-6963).

To address some of the problems noted above, disclosed herein is a“catch and selective release” type method developed for MIDA boronatehydrolysis. Specifically, a THF solution of a MIDA boronate(reactivity=OFF) is mixed with solid-supported ammonium hydroxidereagent (such as Amberlyst A26(OH); see T. M. Morwick, J. Comb. Chem.2006, 8, 649-651) to promote the MIDA hydrolysis. At this point, boththe cleaved MIDA ligand (likely in the form of MIDA²⁻Na⁺ ₂) and theboronic acid (likely in the form of the corresponding anionicboron-‘ate’ complex; see D. G. Hall, J. Tailor, M. Gravel, Angew. Chem.Int. Ed. 1999, 38, 3064-3067) remain trapped in the resin (the “catch”).It was determined that subsequent treatment with a THF solution of AcOH(see M. G. Siegel, P. J. Hahn, B. A. Dressman, J. E. Fritz, J. R.Grunwell, S. W. Kaldor, Tetrahedron Lett. 1997, 38, 3357-3360) resultsin “selective release” of only the boronic acid (reactivity=ON), whilethe cleaved MIDA ligand conveniently remains trapped in the resin underthese mildly acidic conditions. Transferring this THF/AcOH/boronic acidsolution to a new vial containing K₂CO₃, 4 Å molecular sieves, andCelite®, followed by bubbling argon through the mixture and filtrationyields a neutralized, mostly anhydrous, and deoxygenated solution offreshly-prepared boronic acid in THF, ready for the next cross-couplingreaction.

One aspect of the invention relates to the deprotection of a MIDAboronate, comprising the step of contacting a solution comprising theMIDA boronate and a solvent with a solid-supported ammonium hydroxidereagent, thereby deprotecting the MIDA boronate and forming a boronicacid and a MIDA.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solvent comprises THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent binds the MIDA.

In certain embodiments, the present invention relates to any one of theaforementioned methods, further comprising the steps of removing thesolvent by filtration, thereby leaving the boronic acid and MIDA ligandtrapped inside the solid-supported ammonium hydroxide reagent; andadding additional solvent.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the additional solvent is THF.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is washed with an organic solution comprising an organic solventand a mild or strong acid in a quantity greater than that needed toneutralize the solid-supported ammonium hydroxide reagent, therebyeluting the boronic acid.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is washed with a THF solution comprising a mild or strong acid,thereby eluting the boronic acid.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is washed with a THF solution comprising acetic acid, therebyeluting the boronic acid.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the eluted boronic acid is treated withbase to neutralize the acid (e.g., acetic acid).

In certain embodiments, the present invention relates to any one of thein aforementioned methods, wherein the base is potassium carbonate.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is washed with a 1,4-dioxane solution comprising hydrochloricacid, thereby eluting the boronic acid.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is a strong base anion exchange resin, e.g., Amberlite IRA-400(OH⁻ form), Amberlite IRA 420 (OH⁻ form), Amberlite IRA 410 (OH⁻ form),Amberlite IRN-150, Amberlite IRA 900 (OH⁻ form), Amberlite IRA 904 (OH⁻form), Amberlite IRA 910 (OH⁻ form), Amberlite A5836, Amberlyst A26 (OH⁻form), Ambersep 900, Dowex-1 (OH⁻ form), Dowex-3 (OH⁻ form), Dowex 1-X4(OH⁻ form), Dowex 1-I 9880, Dowex 1-I0131, Dowex 550 A (OH⁻ form), orAmberjet 4400.

In certain embodiments, the present invention relates to any one of theaforementioned methods, wherein the solid-supported ammonium hydroxidereagent is a strong base, type 1, anionic, macroreticular polymericresin based on crosslinked styrene divinylbenzene copolymer containingquaternary ammonium groups, e.g., Amberlyst A26 (OH⁻ form) (Rohm andHaas, Philadelphia, Pa.).

An aspect of the invention relates to a method of deprotecting a MIDAboronate, comprising the step of contacting a solution comprising theMIDA boronate and a solvent with an aqueous solution of NaOH, therebydeprotecting the MIDA boronate and forming a boronic acid and free MIDAligand. This method is particularly useful in connection withacid-sensitive substrates (boronic acids) because it does not includeexposure to acid for elution from a solid support.

Because water will be removed in subsequent steps, it is generallydesirable to limit the volume of the aqueous component (solution ofNaOH) introduced into the system to a relatively small amount, e.g.,about 25-33 percent of the volume of the solution comprising the MIDAboronate and its solvent.

In certain embodiments, the present invention relates to theaforementioned method, wherein the solvent comprises THF.

In certain embodiments, the present invention relates to theaforementioned method, wherein the solvent is THF. In one embodiment,the THF is dry and deoxygenated.

In certain embodiments, the present invention relates to theaforementioned method, further comprising the steps of adding diethylether, thereby generating a biphasic mixture comprising an organic phasecomprising the boronic acid and free MIDA ligand, and an aqueous phase;and separating the organic phase comprising the boronic acid and freeMIDA ligand from the aqueous phase. The step of adding diethyl ether canoptionally include adding a reagent effective for quenching thereaction. In one embodiment, the reagent effective for quenching thereaction is a phosphate buffer. Again, because water will be removed insubsequent steps, it is generally desirable to limit the total amount ofwater introduced into the system to a relatively small amount, e.g.,about 25-33 percent of the volume of the combined solution comprisingthe MIDA boronate, its organic solvent, and the aqueous solution ofNaOH. In one embodiment, the phosphate buffer is added in an amountapproximately equal to the volume of the aqueous solution of NaOH.

In certain embodiments, the present invention relates to any one of theaforementioned methods, further comprising the step of contacting theorganic phase with one or more drying agents selected from the groupconsisting of magnesium sulfate, diatomaceous earth, and molecularsieves, thereby drying the organic phase comprising the boronic acid andfree MIDA ligand. A diatomaceous earth can be, for example, Celite®(Fluka/Sigma-Aldrich, St. Louis, Mo.; Celite Corp., Lompoc, Calif.).

In certain embodiments, the present invention relates to any one of theaforementioned methods, further comprising the step of deoxygenating thedried organic phase comprising the boronic acid and free MIDA ligand. Inone embodiment, the deoxygenation is accomplished by bubbling dryoxygen-free gas through the organic phase comprising the boronic acidand free MIDA ligand. In one embodiment, the oxygen-free gas is argon.

Automated Small Molecule Synthesizers

With robust and general methods for the purification and deprotection ofMIDA boronates in hand, an apparatus with the capacity forfully-automated synthesis of small molecules via ICC (FIG. 1D) wasdesigned and built. In certain embodiments, this apparatus is comprisedof three modules, each designed to promote a deprotection (D),cross-coupling (CC), or purification (P) step required to execute theICC scheme depicted in FIG. 1B. In certain embodiments, all materialsare transferred between modules as solutions manipulated by a series ofmain syringe pumps (e.g., eight) coordinated with a suite of switchablevalves (J-KEM Scientific). In certain embodiments, all of the syringepumps are driven by a computer running a custom-made software program.One embodiment of the machine is depicted in FIG. 1D; additional detailsregarding this machine are provided in the Exemplification below.

Reaction System Design.

In certain embodiments, the cross-coupling reactions are run inpolypropylene tubes purchased from Luknova, item #FC003012. Thedimensions of the tube are 21 mm×120 mm (ID×length). The bottom of thetube is fitted with a 21 mm diameter×4 mm tall frit. On top of this fritis secured via metal wire a 13 mm diameter×4 mm tall frit. On top of thefrit is placed a large stir bar containing a rare-earth magnet (BigScience Inc., SBM-1508-REH). The bottom of the tube is accessed througha male Luer tip, while the top of the tube is sealed with an air-tight,threaded cap containing a female Luer port. The tube holds a solventvolume of up to 25 mL. The tubes are placed in an aluminum heating blockthat was custom fabricated. The heating block holds up to nine reactiontubes. The tubes are held 3 cm above the surface of the stir plate,where the bottom 4 cm of the tube is jacketed by the heating block. Thetubing to access the bottom of the reaction tube goes through a hole inthe side of the block near the bottom.

The use of a polypropylene tube appears to be important in simplifyingthe engineering of the reaction tube. Specifically, the material is agood insulator such that only the portion that is jacketed by theheating block becomes hot. When the heating block is heated to 60° C.,the reaction solution reaches 60° C. within several minutes. However,the portion of the tube that is not jacketed remains at roomtemperature, acting as a condenser, and thus the vapor above the solventremains at room temperature. When other materials such as glass wereused, the portion of the tube above the heating block became hot and thesolution quickly evaporated. Thus, if glass were to be used instead ofpolypropylene, there would need to be an additional cooling element inorder to keep the solution from escaping.

In certain embodiments of the system the tubes in the reaction block arestirred constantly, regardless of whether there is solution inside thetube. This keeps the system simple since the stir plate does not need tobe turned on or off, and further, the start and stop times of thereactions within the block do not need to be coordinated. However,during prolonged stirring the stir bar in the tube acts as a mortar andthe frit as a pestle, such that the base becomes finely ground into thepores of the frit. Further, the stir bar may be damaging the top of thefrit even in the absence of base. In these situations it becomes nearlyimpossible to withdraw solutions through the frit since the top surfaceof the frit is clogged and/or damaged. To overcome this limitation areaction tube was designed to contain two frits of different sizes (FIG.8). This way the stir bar only contacts the smaller top frit, and evenif the top surface of this frit is damaged or clogged, the solution canbe withdrawn through the sides of the small frit or through the spacesopen only to the larger frit. The wire is necessary to secure the topfrit so that it does not rotate sideways during the reaction to becomeperpendicular to the larger frit. In certain embodiments, a single fritcould be fabricated to have a shape similar to the combined frits.

Purification System Design.

The chromatographic properties of MIDA boronates that enable the simplepurification approach are discussed above. Below is described how theengineering of the system supports the catch-and-release chromatographyand precipitation-based purification.

Because diluting the crude THF reaction solution with hexanes will causeimmediate precipitation of the MIDA boronate product, the mixing of THFand hexanes must occur in a container of sufficient volume to hold theprecipitated product. The solvents must also be thoroughly mixed so thatthe solution is homogeneous. Finally, the addition of hexanes to the THFsolution also causes reaction byproducts to become insoluble. In somecases these byproducts are sticky or form a very fine precipitate thatcan clog a frit. In certain embodiments, a custom designed precipitationchamber in which to mix the crude THF solution and hexanes can be used(FIG. 9). In certain embodiments, the precipitation chamber containsCelite® which scavenges the sticky impurities that precipitate and keepsthis material distributed throughout the Celite® so it does not clog thefrit. In certain embodiments, a stir bar in the chamber ensures propermixing. However, it was observed that if the stir bar stirs continuouslyfor several hours, the Celite® in the tube becomes so finely ground thatit can pass through the frit and clog downstream processes. To solvethis problem, after the precipitation chamber is filled with solvent andmixed, the solvent is withdrawn so that the stir bar becomes imbedded inthe dry Celite® and does not stir. In other words, the stir bar can bemade to stir only when it is needed—i.e., when there is solvent in theprecipitation chamber—and, therefore, this process does not requireturning the stir plate on/off or coordinating the stirring of otherprocesses that use the same stir plate. In certain embodiments,3-aminopropyl-functionalized silica gel is placed in the precipitationchamber to scavenge palladium from a crude reaction solution.

In certain embodiments, the precipitation chamber is first filled withhexanes via the auxiliary pump (thus wetting the SiO₂ column withhexanes in the process). Then, a THF reaction solution is added from thetop. The main pump does not handle both the hexanes and the THF solutionbecause residual hexanes in the syringe might cause the MIDA is boronateproduct to precipitate in the syringe. Further, the main pump does notwithdraw the waste THF:hexanes solution for this reason and to reducecontamination. Thus, once the solvents are mixed in the precipitationchamber, the solution is withdrawn by the auxiliary pump through theSiO₂ column and then sent to waste. Next, Et₂O with MeOH is added to theprecipitation chamber, mixed, and then withdrawn through the SiO₂ columnand sent to waste. This process is repeated with Et₂O. At this point thepure MIDA boronate product remains as a precipitate in the precipitationchamber or is at the very top of the SiO₂ column. The auxiliary pumpthen injects THF through the bottom of the SiO₂ column, out through thetop and through to the precipitation chamber. Thus, it does not matterif the MIDA boronate product is initially in the SiO₂ column or in theprecipitation chamber, since it will be dissolved in THF at eitherlocation. This system reduces the amount of THF used to elute/dissolvethe product, allowing the solution to be used in the next reactionwithout further concentration. After the THF has been mixed in theprecipitation chamber for about 30 minutes, it is withdrawn by the mainpump through the 3-way union without again passing through the SiO₂column.

Some important features of this setup are: the precipitation eventoccurs in a mixing chamber; the mixing chamber contains scavengers(Celite® and functionalized silica gel); the stirring can be controlledin a simple way; the precipitation chamber and SiO₂ column are spatiallyseparated; and the configuration of pumps allows solvents to be addedand withdrawn at various junctions throughout the process.

Software.

The software that controls the machine can be described as having threelevels of complexity: the basic level, the functional level, and thedeveloped level. The basic level represents essentially the combinationof 1's and 0's that can be sent to the equipment to move the syringepumps and valves. The functional level represents the simplest commandsto move the equipment that could be understood by a person looking atthe computer code. The developed level represents software specificallytailored to the automated synthesis machine that can be used bynon-experts to modify how the synthesis is performed. The basic level isinherent to the equipment manufactured by Kloehn. The functional levelcame from source code provided by J-KEM when the equipment waspurchased. The functional level is source code written in VB.NET thatpackages the commands of the basic level into easily executedsubroutines. The developed level was custom designed and written inVB.NET based on the source code provided in the functional level.

More specifically, the machine is composed of a number of syringe pumpsand valves that are OEM parts from Kloehn. The syringe pumps and valveswere repackaged and sold by J-KEM as a custom piece of hardware. Theequipment is controlled via a RS-485 serial port that sends and receivessimple text string commands written in machine language specific to theKloehn parts. Thus, on the simplest level the machine can be controlledby sending simple text strings such as “/xR”, but these commands areunintelligible to anyone using the equipment. The equipment shipped fromJ-KEM came with source code written in VB.NET that provides subroutinesto move the syringe pump to specific positions, change valves tospecific positions, control the rate at which the syringe moves, andopen/close solenoid valves. The code that was developed uses thesubroutines from the code provided by J-KEM. The source code from J-KEMcould be removed and the software could communicate with the equipmentdirectly without losing functionality. However, the source code fromJ-KEM would not be by itself enough to run an automated synthesis. Thus,it was necessary to create software to enable the development of theautomated synthesis machine. Details regarding certain embodiments ofthe software can be found in the Exemplification.

Automated Synthesizers.

One aspect of the invention relates to an automated small moleculesynthesizer comprising: (a) a deprotection module, in fluidcommunication with (b) a drying and degassing module, in fluidcommunication with (c) a reaction module, in fluid communication with(d) a purification module; at least one pump which can move liquid fromone module to another; and a computer equipped with software; whereinall of the modules are under the control of the computer.

In one embodiment, the deprotection module comprises immobilized orsolid-supported base, e.g., NaOH, as described herein. In oneembodiment, the deprotection module is constructed and arranged so as toperform deprotection with aqueous base, e.g., aqueous NaOH, as describedherein.

In one embodiment, the purification module comprises a combined (or“hybrid”) precipitation and catch-and-release module.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thedeprotection module comprises a deprotection chamber which comprises afirst opening at the top of the chamber, a second opening at the bottomof the chamber, a first frit covering the second opening, and asolid-supported ammonium hydroxide reagent.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thedeprotection chamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber is a polypropylenecylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber has a length of betweenabout 100 mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber has a length of about 120mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber has an interior diameter ofbetween about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber has an interior diameter ofabout 21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the deprotection chamber has a volume of about 25mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thesolid-supported ammonium hydroxide reagent is a strong base anionexchange resin, e.g., Amberlite IRA-400 (OH⁻ form), Amberlite IRA 420(OH⁻ form), Amberlite IRA 410 (OH⁻ form), Amberlite IRN-150, AmberliteIRA 900 (OH⁻ form), Amberlite IRA 904 (OH⁻ form), Amberlite IRA 910 (OH⁻form), Amberlite A5836, Amberlyst A26 (OH⁻ form), Ambersep 900, Dowex-1(OH⁻ form), Dowex-3 (OH⁻ form), Dowex 1-X4 (OH⁻ form), Dowex 1-I 9880,Dowex 1-I0131, Dowex 550 A (OH⁻ form), or Amberjet 4400.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thesolid-supported ammonium hydroxide reagent is a strong base, type 1,anionic, macroreticular polymeric resin based on crosslinked styrenedivinylbenzene copolymer containing quaternary ammonium groups, e.g.,Amberlyst A26 (OH⁻ form).

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thedeprotection module further comprises a source of gas; wherein thesource of gas can be placed in fluid communication with the deprotectingchamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingand degassing module comprises a combined drying and degassing chamberwhich comprises a first opening at the top of the combined drying anddegassing chamber, a second opening at the bottom of the combined dryingand degassing chamber, a first frit covering the second opening, and aplunger; and the drying and degassing module is in fluid communicationwith the deprotection module.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecombined drying and degassing chamber further comprises a diatomaceousearth, e.g., Celite®.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecombined drying and degassing chamber further comprises activatedmolecular sieves.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein themolecular sieves are 4 angstrom, 8-12 mesh.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecombined drying and degassing chamber further comprises potassiumcarbonate.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the fluidcommunication is a result of the connection of the second opening of thedeprotection chamber to the second opening of the combined drying anddegassing chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecombined drying and degassing chamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber is apolypropylene cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber has alength of between about 100 mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber has alength of about 120 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber has aninterior diameter of between about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber has aninterior diameter of about 21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the combined drying and degassing chamber has avolume of about 25 mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga source of argon.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the argoncan be placed in fluid communication with the combined drying anddegassing chamber to sparge the contents of the combined drying anddegassing chamber; and the plunger prevents solids from lifting duringsparging.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstopening of the combined drying and degassing chamber is vented to aninert gas atmosphere which is maintained near atmospheric pressure viaventing through an oil-filled bubbler.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the argoncan be placed in fluid communication with the combined drying anddegassing chamber through the second opening of the tube of the combineddrying and degassing chamber while the first opening of the tube of thecombined drying and degassing chamber is vented to an inert gasatmosphere.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingand degassing module comprises a drying chamber and a degassing chamber;the drying chamber comprises a first opening at the top of the dryingchamber, a second opening at the bottom of the drying chamber, a firstfrit covering the second opening, and a plunger; the degassing chambercomprises a first opening at the top of the degassing chamber and asecond opening at the bottom of the degassing chamber; the dryingchamber is in fluid communication with the degassing chamber; and thedegassing chamber is in fluid communication with the deprotectionmodule.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingchamber further comprises a diatomaceous earth, such as Celite®.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingchamber further comprises activated molecular sieves.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein themolecular sieves are 4 angstrom, 8-12 mesh.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingchamber further comprises potassium carbonate.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the fluidcommunication between the drying chamber and the degassing chamber is aresult of the connection of the second opening of the drying chamber tothe second opening of the degassing chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the fluidcommunication between the drying and degassing module and the degassingchamber is a result of the connection of the second opening of thedegassing chamber to the deprotection module.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the dryingchamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber is a polypropylene cylindricaltube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber has a length of between about 100mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber has a length of about 120 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber has an interior diameter ofbetween about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber has an interior diameter of about21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the drying chamber has a volume of about 25 mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thedegassing chamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber is a polypropylene cylindricaltube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber has a length of between about100 mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber has a length of about 120 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber has an interior diameter ofbetween about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber has an interior diameter ofabout 21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the degassing chamber has a volume of about 25 mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga source of argon.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the argoncan be placed in fluid communication with the degassing chamber tosparge the contents of the degassing chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstopening of the degassing chamber is vented to an inert gas atmospherewhich is maintained near atmospheric pressure via venting through anoil-filled bubbler.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the argoncan be placed in fluid communication with the degassing chamber throughthe second opening of the tube of the degassing chamber while the firstopening of the tube of the degassing chamber is vented to an inert gasatmosphere.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction module comprises a reaction chamber which comprises a firstopening at the top of the reaction chamber, a second opening at thebottom of the reaction chamber, a first frit covering the secondopening, and a stir bar; wherein the reaction module is in fluidcommunication with the drying and degassing module.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction module comprises a third opening at the top of the reactionchamber through which a liquid can be added to the reaction chamberwithout contacting the sidewalls or the bottom of the reaction chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstopening of reaction chamber is vented to an inert atmosphere.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstopening of the reaction chamber is fitted with a fritted tube to preventfine solids from escaping from the reaction chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstopening of the reaction chamber is vented to an inert atmospheremaintained near atmospheric pressure via venting through an oil-filledbubbler.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein both thesecond opening and third opening of the reaction chamber are in fluidcommunication with the second opening of the drying and degassingchamber at the same time.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction chamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber is a polypropylene cylindricaltube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber has a length of between about100 mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber has a length of about 120 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber has an interior diameter ofbetween about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber has an interior diameter ofabout 21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the reaction chamber has a volume of about 25 mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction chamber further comprises a second frit between the stir barand the first frit; wherein the second frit is smaller than the firstfrit.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the secondfrit and the first frit are held together with a wire, to prevent thesecond frit from turning perpendicular to the first frit.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstfrit is disc-shaped; the first frit has a diameter of between about 18mm and about 24 mm; and the first frit has a height between about 2 mmand about 6 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstfrit is disc-shaped; the first frit has a diameter of about 21 mm; andthe first frit has a height of about 4 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the secondfrit is disc-shaped; the second frit has a diameter between about 16 mmand about 10 mm; and the second frit has a height between about 2 mm andabout 6 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the secondfrit is disc-shaped; the second frit has a diameter of about 13 mm; andthe second frit has a height of about 4 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the shapeof the first frit is that of a first disc on top of a second disc;wherein the diameter of the first disc is smaller than the diameter ofthe second disc.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the shapeof the first frit prevents solids from passing through the secondopening of the reaction chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction module further comprises a stir plate which turns the stir bar.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction module further comprises a heating block which can heat thecontents of the reaction chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein at least aportion of the reaction chamber is jacketed by the heating block.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction chamber further comprises a transition metal salt.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thetransition metal salt is adsorbed onto a solid.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thetransition metal salt is palladium acetate.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the solidonto which the transition metal salt is adsorbed is cesium carbonate.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction chamber further comprises a phosphine ligand.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thephosphine ligand is adsorbed onto a solid.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thephosphine ligand is S-Phos(2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl).

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecatalyst is derived from an air-stable palladium precatalyst.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the solidonto which the phosphine ligand is adsorbed is cesium carbonate.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction chamber further comprises a base.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the basein the reaction chamber is potassium hydroxide.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction run in the reaction chamber is a cross-coupling reaction.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction run in the reaction chamber is selected from the groupconsisting of a Suzuki-Miyaura coupling, an oxidation, a Swernoxidation, a “Jones reagents” oxidation, a reduction, an Evans' aldolreaction, an HWE olefination, a Takai olefination, an alcoholsilylation, a desilylation, a p-methoxybenzylation, an iodination, aNegishi cross-coupling, a Heck coupling, a Miyaura borylation, a Stillecoupling, and a Sonogashira coupling.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thereaction run in the reaction chamber is a Suzuki-Miyaura coupling.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thechemical reaction run in the reaction chamber comprises the step ofcontacting a MIDA boronate with a reagent, wherein the MIDA boronatecomprises a boron having an sp³ hybridization, a MIDA protecting groupbonded to the boron, and an organic group bonded to the boron through aboron-carbon bond; the organic group is chemically transformed, and theboron is not chemically transformed.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the MIDAboronate is represented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of a hydrogen group and anorganic group.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein R²¹, R²²,R²³ and R²⁴ are hydrogen.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module comprises a precipitation chamber and a silicacolumn; the precipitation chamber comprises a first opening at the topof the precipitation chamber, a second opening at the bottom of theprecipitation chamber, a first frit covering the second opening, a stirbar, and a diatomaceous earth (such as Celite®); and the silica columncomprises a first opening at the top of the column, a second opening atthe bottom of the column, a second frit covering the top opening of thecolumn, a third frit covering the bottom opening of the column, andsilica; wherein the purification module is in fluid communication withthe reaction module.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theprecipitation chamber comprises a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber is a polypropylenecylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber has a length of betweenabout 100 mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber has a length of about 120mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber has an interior diameterof between about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber has an interior diameterof about 21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the precipitation chamber has a volume of about 25mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theprecipitation chamber further comprises a resin which scavenges metals.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theprecipitation chamber further comprises activated charcoal.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module further comprises a stir plate which turns the stirbar.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thediatomaceous earth (e.g., Celite®) in the precipitation chamber preventsthe stir bar from turning if there is no solvent in the precipitationchamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the firstfrit of the precipitation chamber keeps the diatomaceous earth in theprecipitation chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the silicacolumn is a cylindrical tube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column is a polypropylene cylindricaltube.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column has a length of between about 100mm and 140 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column has a length of about 120 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column has an interior diameter ofbetween about 18 mm and about 24 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column has an interior diameter of about21 mm.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thecylindrical tube of the silica column has a volume of about 25 mL.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the secondfrit of the silica column and third frit of the silica column keep thesilica in the silica column.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the silicais functionalized with amino groups.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module further comprises an auxiliary pump.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga solvent reservoir containing hexane.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module is in fluid communication with the reservoir ofhexane; and the auxiliary pump provides hexane from the reservoir to theprecipitation chamber by passing the hexane into the silica columnthrough the second opening of the silica column, out of the silicacolumn through the first opening of the silica column, and into theprecipitation chamber through the second opening of the precipitationchamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theauxiliary pump can remove the hexane by passing the hexane into thesecond opening of the precipitation chamber, through the first openingof the silica column and out the second opening of the silica column.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga solvent reservoir containing diethyl ether.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module is in fluid communication with the reservoir ofdiethyl ether; and the auxiliary pump provides diethyl ether from thereservoir to the precipitation chamber by passing the diethyl ether intothe silica column through the second opening of the silica column, outof the silica column through the first opening of the silica column, andinto the precipitation chamber through the second opening of theprecipitation chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theauxiliary pump can remove the diethyl ether by passing the diethyl etherinto the second opening of the precipitation chamber, through the firstopening of the silica column and out the second opening of the silicacolumn.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga solvent reservoir containing diethyl ether containing 1.5% methanol byvolume.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module is in fluid communication with the reservoir ofdiethyl ether containing 1.5% methanol by volume; and the auxiliary pumpprovides diethyl ether containing 1.5% methanol by volume from thereservoir to the precipitation chamber by passing the diethyl ether intothe silica column through the second opening of the silica column, outof the silica column through the first opening of the silica column andinto the precipitation chamber through the second opening of theprecipitation chamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein theauxiliary pump can remove the diethyl ether containing 1.5% methanol byvolume by passing the diethyl ether containing 1.5% methanol by volumeinto the second opening of the precipitation chamber, through the firstopening of the silica column and out the second opening of the silicacolumn.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga waste container; wherein the second opening of the silica column is influid communication with the waste container.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, further comprisinga solvent reservoir containing THF.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein thepurification module is in fluid communication with the reservoir of THF;and the auxiliary pump provides THF from the reservoir to theprecipitation chamber by passing the THF into the silica column throughthe second opening of the silica column, out of the silica columnthrough the first opening of the silica column, and into theprecipitation chamber through the second opening of the precipitationchamber.

In certain embodiments, the present invention relates to any one of theaforementioned automated small molecule synthesizers, wherein the secondopening of the precipitation chamber can be placed in fluidcommunication with the first opening of the silica column via athree-way valve; wherein a first port on the valve is connected to thesecond opening of the precipitation chamber, a second port on the valveis connected to the first opening of the silica column, and a third porton the valve can be used to withdraw a solution from the precipitationchamber without passing the solution through the silica column.

Another aspect of the invention relates to any one of the aforementionedautomated small molecule synthesizers, comprising: one or moredeprotection modules; one or more drying and degassing modules; one ormore reaction modules; one or more purification modules; at least onepump which can move liquid from one module to another; and a computerequipped with software; wherein all of the modules are under the controlof the computer.

Another aspect of the invention relates to any one of the aforementionedautomated small molecule synthesizers comprising: a plurality ofdeprotection modules; a plurality of drying and degassing modules; aplurality of reaction modules; a plurality of purification modules; atleast one pump which can move liquid from one module to another; and acomputer equipped with software; wherein all of the modules are underthe control of the computer.

Alternative Embodiment Using Aqueous Deprotection Module.

The aqueous deprotection module consists of equipment necessary tocomplete a solution-phase aqueous base-mediated MIDA boronatedeprotection, a separation of the resulting biphasic mixture, apredrying and drying of the organic layer (e.g., ethereal solution ofthe boronic acid), and a deoxygenation/concentration of the driedorganic layer in preparation for a cross-coupling.

Specifically, two syringe pumps and an argon solenoid are utilized inthe new deprotection module (FIG. 9). The primary syringe pump, which isused for the majority of liquid handling during an entire sequence,handles organic solvents and solutions. A dedicated aqueous syringe pumpis utilized to handle all aqueous reagents (water, 0.5 M, pH=6,potassium phosphate buffer, and 50% saturated sodium chloride). Thisisolation of aqueous solutions to a dedicated syringe pump minimizeswater contamination throughout the rest of the machine. The argonsolenoid is used to deliver a flow of dry argon for agitating thedeprotection and for concentrating and deoxygenating the final solutionof boronic acid.

For example, deprotection at the beginning of a multistep sequencebegins with solid MIDA boronate (1 mmol, 1 equiv) and solid sodiumhydroxide (3 mmol, 3 equivs) in a 12-g Luknova cartridge. The primarypump delivers dry deoxygenated THF (10 mL, 0.1 M) to dissolve the MIDAboronate. The aqueous pump then delivers deionized water (3 mL, 0.33 M),creating a biphasic mixture, to dissolve the sodium hydroxide. A flow ofdry argon is then delivered (in short 0.5-2 second pulses) from thebottom of the tube, bubbling through and agitating the biphasic mixturefor 10 minutes at which time the deprotection is complete. Then,simultaneously the aqueous pump delivers phosphate buffer (3 mL) toquench the reaction, and the primary pump delivers diethyl ether (5 mL)to prepare for the separation. The aqueous pump then agitates thequenched reaction with several injections of atmospheric air. Theaqueous pump then aspirates the biphasic reaction mixture, pauses toallow full separation, and returns the remaining organic layer to thedeprotection tube. The aqueous layer is injected to waste and theaqueous pump delivers 50% saturated sodium chloride (3 mL) to thedeprotection tube and agitates the mixture with several injections ofair. Again, the aqueous pump aspirates the biphasic mixtures, pauses toallow full separation, and returns the organic layer to the deprotectiontube. The aqueous layer is injected to waste.

This separation has been shown to be reproducible in the production ofaqueous layer volumes. On a 1 mmol scale the first aqueous layer is 6.0mL (±0.1 mL). On a 0.66 mmol scale the first aqueous layer is 6.2 mL(±0.1 mL). On a 0.33 mmol scale the first aqueous layer is 6.4 mL (±0.1mL). The aqueous salt volume has been shown to be 3.8 mL (±0.1 mL)regardless of reaction scale.

Subsequent deprotections begin with the purified MIDA boronate as asolution in THF (from the automated purification) being injected into anew deprotection tube containing sodium hydroxide. The remainder of thedeprotection proceeds as described above. For these downstreamdeprotections the only difference in experimental setup is the amount ofsodium hydroxide used (the stoichiometry is always 3 equivalents withrespect to the MIDA boronate). The solvent and reagent volumes remainthe same and are as outlined above. The separation volumes have shown ascale dependency and are as outlined above. The remaining manipulations(predrying, drying, and deoxygenating and concentration) proceed asdescribed below. The relative volumes and quantities of solvents andreagents are independent of reaction scale.

Predrying of the still-wet organic layer (ethereal solution of boronicacid) removes the bulk of the remaining water. In one embodiment, thepredrying tube is a 12-g Luknova cartridge containing a mixture ofCelite® (800 mg) and anhydrous magnesium sulfate (2.1 g). A 5-mLpolypropylene syringe plunger is placed on top of the solid mixture. Thetwo solids are mixed intimately to prevent clumping of the magnesiumsulfate clathrate. Additionally, the syringe plunger prevents movementof the solids up the tube as liquids are injected. To begin thepredrying step, the primary pump delivers 5 mL of dry deoxygenated THFto the predrying tube. It has been shown that the solids adsorbapproximately 5 mL of THF during this process, so wetting the solidswith clean THF prevents loss of volume. Next, the primary pump transfersthe organic layer from the deprotection tube into the predrying tube.The solution is passed over the solid mixture by repeatedaspiration/injection (rate=15 mL/min) via the primary pump. In total,the solution is agitated in this manner 20 times. At this point the bulkwater has been removed from the solution of boronic acid.

Drying of the ethereal boronic acid solution is required to remove theremaining residual water. In one embodiment, the drying tube is a 12-gLuknova cartridge containing a layer of Celite® (300 mg) topped withactivated molecular sieves (4 Å, −325 mesh, 3.6 g). A 5-mL polypropylenesyringe plunger is placed on top of these solids. The bottom layer ofCelite® prevents clogging of the tube frit by the fine molecular sieves.The syringe plunger prevents movement of solids as described above. Tobegin the drying step, the primary pump delivers 5 mL of drydeoxygenated THF to the predrying tube. As described above, thisprevents loss of volume. Next, the primary pump transfers the predriedsolution of boronic acid from the predrying tube to the drying tube.Similar to the agitation method described above, the solution is passedover the solids by repeated aspiration/injection (rate=5 mL/min) via theprimary pump. In total, the solution is agitated in this manner 12times. The rate of aspiration during the drying step achievesappropriate aspiration and thereby thorough agitation. Specifically, aslow aspiration rate of about 5 mL/min efficiently aspirates the boronicacid solution through the layer of molecular sieves. Faster rates resultin the build-up of vacuum that is dissipated by solvent evaporationrather than solution aspiration; boronic acid solution is notefficiently passed over the molecular sieves and remains wet. At thispoint the boronic acid solution has been thoroughly dried.

Deoxygenating the boronic acid solution is required in preparation ofthe cross-coupling reaction. Specifically, the solution needs to bedeoxygenated for the coupling to proceed productively. Additionally,concentration of the solution is useful to remove any diethyl ether thatis still present from the deprotection workup, as well as to maintain aworkable volume for the cross-coupling reaction. Workable relativevolumes for the coupling reactions have been determined to be 9 mL ofboronic acid solution for all couplings, regardless of reaction scale.As an exception, the final reaction in a sequence requires a boronicacid solution of 2 mL. In one embodiment, the concentration tube is anempty 12-g Luknova cartridge. To begin the deoxygenating/concentrationstep, the primary pump transfers the dry boronic acid solution to theconcentration tube. Then, dry argon is bubbled through the solution tosimultaneously deoxygenate and concentrate. The argon flow begins withshort 0.5 second pulses and these pulses become progressively longerover the course of 3 minutes, at which point the argon flow remains oncontinuously. This concentration process has been shown to reduce volumeat an approximate rate of 0.1 mL/min. Before concentration, the volumeis 18 mL and, therefore, approximately 90 minutes of argon flow reducesthe volume to 9 mL. The resulting dry, deoxygenated, concentratedsolution of boronic acid is suitable for addition to an anhydrouscross-coupling reaction.

The aqueous deprotection module represents a robust and predictablemethod for the automation of MIDA boronate deprotection reactions. Thesetypes of aqueous conditions are known to work for many sensitive boronicacids in the context of non-automated synthesis, and, as such, thisautomated deprotection is expected to work reliably for a wide range ofsensitive substrates. This aqueous deprotection module, however, differsfrom previously reported methods of MIDA boronate deprotection inseveral ways. The changes employed in the automated process relative tothe procedure utilized in published solution-phase reactions includeargon flow agitation and argon sparging deoxygenation/concentration, athree-step drying strategy, the minimization and use of specific solventvolumes, and controlled slow-rate aspiration for liquid handling.

In a non-automated MIDA boronate deprotection, agitation of the biphasicreaction is achieved with conventional stirring (magnetic stir bar andstirring plate). The aqueous deprotection module of the inventionutilizes argon gas flow to agitate the deprotection reaction. As argonis passed through the frit of the deprotection tube, the resultingbubbles provide highly efficient agitation of the biphasic system. Theagitation is sufficient to achieve full conversion at room temperaturein 10 min (similar to non-automated conditions) without the use of astir plate. Furthermore, the aqueous deprotection module uses argon flowto sparge and concentrate the boronic acid solution. In non-automatedsyntheses with stable boronic acids, the acid is typically isolated as asolid and submitted to a cross-coupling in the presence of deoxygenatedsolvent. In the case of unstable boronic acids, the acid is typicallynot isolated, but concentrated to some small volume by iterativeconcentrations from deoxygenated solvent. Use of argon sparging and gasflow concentration simultaneously in the automated system deoxygenatesthe boronic acid solution and concentrates it. This provides acoupling-ready boronic acid solution without the need to isolate apotentially unstable boronic acid.

Drying of the boronic acid solution for a non-automated synthesistypically involves drying over an anhydrous drying reagent, filtrationthrough Celite®, and subsequent washing of the drying reagent. The useof excess drying reagent can insure complete drying, and the use ofcopious solvent volumes can insure quantitative recovery. This increasedsolvent volume presents a challenge in the context of automation. Thatis, all the excess volume accumulated in the automated process wouldneed to be concentrated downstream. In order to minimize theaccumulation of solvent, which is closely connected to the dryingprocess, the automated system utilizes a cooperative three-step dryingstrategy and specific solvent volumes. The first of the three steps is a50% saturated sodium chloride extraction of the organic phase of thedeprotection reaction. This removes some bulk water from the organicphase and, as described above, does so with reproducible specificvolumes. The second of the three steps is the predrying of the boronicacid solution over anhydrous magnesium sulfate, which removes more bulkwater from the solution. The final step is the drying over molecularsieves, which removes the remaining residual water. As described above,each step uses specific predetermined solvent volumes to maintainminimized, yet reproducible solvent accumulation. This process, coupledwith the use of predetermined, minimized quantities of drying agents,maximizes drying and substrate recovery while minimizing solventaccumulation.

Another key difference between previously reported methods fornon-automated syntheses and the automated system of the invention is theuse of controlled slow-rate aspiration for liquid handling. Thedecreased aspiration rate during the drying step, as described above,has enabled efficient handling of liquids. Specifically, aspirationrates above 2 mL/min during the drying step cause a build-up of vacuumin the primary syringe pump. The vacuum is then relieved by theevaporation of solvent. As a result, the boronic acid solution is notfully aspirated and is not efficiently dried over the molecular sieves.Using a decreased aspiration rate minimizes the build-up of vacuum andallows for full aspiration of the boronic acid solution. This slow-rateaspiration approach has also been applied to the aspiration of crudecross-coupling reaction mixtures.

Specific volumes and amounts disclosed above can, of course, be scaledup or down to suit larger or smaller overall scale automated machines,respectively, provided that the scaled volumes and scaled amounts remainproportional to one another.

An Exemplary Automated Coupling Cycle.

An example of one complete cycle of automated coupling proceeds asfollows.

Step 1. In the deprotection module, catch and selective release-basedhydrolysis of a MIDA boronate yields a freshly-prepared boronic acid asa solution in THF.

In this example, the deprotection of MIDA-protected organoboronic acidsvia solid-supported ammonium hydroxide reagent proceeds without the useof added bulk water, thereby avoiding the need to remove bulk waterprior to a subsequent anhydrous reactions (such as a cross-coupling). Arange of aqueous deprotection conditions in the context of the automatedsynthesis were tried but the following problems were found:

-   -   The amount of solvent required to extract the boronic acid        product depended on the identity of the boronic acid. Polar        boronic acids required much more solvent. Some boronic acids        were too polar to be effectively extracted.    -   The amount of solvent used in the extraction step would require        an additional evaporation step to obtain a reasonable        concentration.    -   Removing the large amount of water in the organic phase required        very large amounts of drying reagents that became impractical.    -   Completely removing the water introduced in the deprotection        step from the machine was very difficult from an engineering        standpoint. Residual water persisted in the tubing and syringes.

Running the deprotection reaction with solid KOH and anhydrous THF didnot proceed. Running the deprotection reaction with solid KOH and 1%water in THF was not a general solution because the two products of thedeprotection reaction, N-methyliminodiacetic acid bis potassium salt andthe boronate salt (the boronic acid reacts with KOH to produce theanionic boronate species), were both insoluble in THF and aggregated tocause the water and the THF to separate and the KOH to be sequestered,thus stalling the reaction. The use of Amberlyst A26(OH) resin solvedall of these problems. The resin is not anhydrous since it is preparedin water and is shipped damp; one can control the amount of water thatis present based on the volumes of organic solvent that are used to washthe resin. Accordingly, it is possible to produce a free-flowing resinthat contains only enough water to allow the deprotection reaction toproceed, and not so much water that the resulting reaction solutions cannot be easily dried with a small amount of molecular sieves. Further,residual water does not contaminate the equipment since bulk water isnever added to the reactions. The problem of aggregation is solvedbecause the N-methyliminodiacetic acid bis potassium salt produced inthe deprotection reaction becomes trapped within the pores of the resinand does not aggregate with unreacted KOH or water. Often the boronatesalt produced in the deprotection reaction also becomes trapped withinthe pores of the resin. Boronate salts trapped in the resin do notaggregate and do not stall the reaction. Further, this protocol is theonly deprotection condition for MIDA boronates that does not requirestirring. The reaction with Amberlyst A26(OH) proceeds to fullconversion within 60 minutes with periodic air bubbling to mix themixture, thus allowing a large number of deprotection reactions to beperformed in parallel with simple equipment. The mixture (resin and THF)is then treated with dilute acetic acid to convert the boronate salt tothe boronic acid. The very fine, very polar N-methyliminodiacetic acidproduced in this process remains trapped in the Amberlyst resin whichgreatly facilitates filtration of the mixture. (Without the Amberlystresin sequestering the N-metyliminodiacetic acid, the subsequentfiltration step was found to be unreliable.)

Step 2. This boronic acid solution is then transferred to thecross-coupling module where it is added slowly to a stirred reactionmixture containing the next halogen-bearing building block, a palladiumcatalyst, and a solid inorganic base. Conversion of each halide buildingblock is maximized via: (a) using excess boronic acid (˜3 equiv.)relative to halide (1 equiv.); (b) employing slow-addition orslow-release of the boronic acid to help avoid its decomposition in situduring the cross-coupling reaction; and (c) using Buchwald's highlyeffective and air-stable SPhosPd catalyst to maximize the generality,efficiency, and mild nature of the cross-coupling condition (D. M.Knapp, E. P. Gillis J. Am. Chem. Soc. 2009, 131, 6961-6963; and R.Martin S. L. Buchwald Acc. Chem. Res. 2008, 41, pp 1461-1473).

Step 3. The soluble components of the resulting crude reaction mixtureare transferred to the purification module where the MIDA boronateproduct is purified via tandem precipitation and catch-and-releaseprocesses, as described above.

In the automated system the THF:hexane solution, Et₂O with 1.5% MeOH(v/v) solution and Et₂O solution are withdrawn from the top of the SiO₂column and through the bottom under vacuum. This approach is differentfrom standard chromatography in which the solution is pushed through thetop of the column under pressure. Again, the unique elution propertiesof the MIDA boronate hold up under this modification, and thismodification greatly simplifies the engineering of the purificationstep. In the automated system the THF is injected into the bottom of thecolumn and flows out through the top under positive pressure. In thisway the MIDA boronate, which is immobilized near the top of the column,has the least distance to be carried in the THF (has the smallest columnvolume) and thus the amount of THF used to elute the MIDA boronate canbe minimized. It is believed that flowing solvents in oppositedirections at separate times on the same column is not a standardchromatography practice.

This three-step cycle of deprotection, cross-coupling, and purificationis iterated until the final building block coupling step is reached. Tomaximize efficiency, the final coupling reaction in each sequence isperformed via in situ hydrolysis of the final MIDA boronate underaqueous basic conditions. “Slow-release” cross-coupling in this contextcan help maximize the yield of this final coupling reaction. Similar tothe approach used in peptide, oligonucleotide, and oligosaccharidecoupling, if the individual building blocks contain other types ofprotective groups, these are collectively removed using manuallyexecuted deprotection reactions prior to an automated chromatographicpurification of the final product (FIG. 1B).

Preparation of Small Molecule Natural Products

With this novel small molecule synthesis apparatus on the benchtop, itscapacity was tested via the preparation a series of small moleculenatural products, derived from various biosynthetic classes, includingpolyterpenes, fatty acids, and oxidative couplings, in a fully automatedfashion (FIG. 2). Details of selected reactions are provided in theExemplification section below. In the idealized ICC approach, such smallmolecules are constructed using only stereospecific cross-couplingreactions to assemble iteratively collections of building blocks havingall of the required functionality pre-installed in the correctoxidations states and with the required stereochemical relationships(several of the building blocks required for these syntheses arecommercially available).

As the chromophore for vision in all vertebrates, the light-harvestingbacteriorhodopsin complex, and a derivative of a key vitamin inmammalian physiology, the polyterpine-derived natural product retinal isthe focus of extensive investigations in chemistry, biology, physics,and medicine. Thus, fully automated synthetic access to this naturalproduct and many of its derivatives stands to have a widespread impacton all of these areas of research. This natural product wasautomatically synthesized using two coupling cycles to unite buildingblocks BB₄, commercially available BB₅, and BB₆ (FIG. 2A).

The fluorescent lipid, β-parinaric acid is derived from fatty acidbiosynthesis, and has proved to be very useful in a wide range ofstructural and functional studies of lipid bilayer membranes.Demonstrating that the modularity that exists in small molecules evencrosses between biosynthetic classes, the same commercially availablebuilding block BB₅ was used twice in the automated preparation ofβ-parinaric acid. Specifically, three iterated cycles of coupling led tothe automated assembly of building blocks BB₇, BB₅, BB₅, and BB₈ toefficiently prepare this small molecule target (FIG. 2B).

In another example, the neolignan ratanhine (A. Arnone, V. Di Modugno,G. Nasini, O. V. de Pava Gazz. Chim. Ital. 1990, 120, 397-401) is amodular natural product derived from oxidative coupling of commonbuilding blocks, in this case phenylpropanoid building blocks derivedfrom phenylalanine (S. R. Angle, K. D. Turnbull, J. Org. Chem. 1993, 58,5360-5369). Like the other derivatives, this building block-basedbiosynthesis strategy results in substantial modularity. For example,the propenyl fragment present in MIDA boronate BB₉ appears in a widerange of natural products, and this building block is now commerciallyavailable. Ratanhine was automatically synthesized via the sequentialcouplings of BB₉, BB₁₀, BB₁₁, and BB₁₂, followed by a final globaldeprotection of the methoxymethyl (MOM) ethers. This type of globaldeprotection is analogous to that used to deprotect automaticallysynthesized peptides, oligonucleotides, and oligosaccharides. See FIG.2C.

As shown in FIG. 2D, the interesting PK/NRPS-derived natural productcrocacin C will be automatically assembled starting withcommercially-available MIDA boronate BB₁. Two cycles of automatedcouplings with, BB₂ and BB₃ are anticipated to yield the naturalproduct.

It is also proposed that the simple and flexible nature of automated ICCmight enable the rapid preparation of derivatives of a specific smallmolecule with the potential to drive fundamental studies of itsfunction. Specifically, as described above, due to its exceptionalimportance across a broad range of scientific disciplines, retinal hasbeen intensely studied by chemists, biologists, biophysicists, andmolecular engineers. Double bond stereochemistry is intimately linked tothe function of this natural product, and probing these relationshipsrequires efficient access to these stereoisomers in chemically pureform. Moreover, retinal derivatives that are site-selectively labeledwith ¹³C atoms are very valuable for biophysical studies mediated by NMRspectroscopy. It is recognized that the ICC approach could potentiallyaddress both of these synthetic challenges simply by incorporating intothe automated synthesis sequence described above alternative MIDAboronate building blocks having the desired stereochemistry andsite-specific ¹³C labeling pre-installed (FIG. 3A). Specifically, thefully-automated iterative coupling of various combinations of buildingblocks BB₄, (E)-BB₅, (Z)-BB₅, (E)-¹³C₂-BB₅, (Z)-¹³C₂-BB₅, BB₆, and¹³C₂-BB₆ is anticipated to yield six different derivatives of retinal,representing two possible stereoisomers, each ¹³C-labeled in threedistinct patterns (FIG. 3B).

Automated ICC also has the theoretical capacity to substantiallyaccelerate the preparation of many derivatives of biologically activesmall molecule natural products with the goal of improving theirpotential for medicinal applications. As a specific example, ratanhinerepresents just one member of a very large family of neolignan naturalproducts. Many of these natural products demonstrate interestingantifungal, antileishmanial, antiangiogenic, antirheumatic, antitumoral,and/or hypolipidemic properties, and several have been used as leadcompounds for the development of new medicines (S. Apers, A. Vlietinck,L. Pieters, Phytochem. Rev. 2003, 2, 201-217). To explore the potentialof automated ICC to prepare structural analogs of this family of naturalproducts, four sets of building blocks representing the varioussubstructures that commonly appear in many of these compounds have beenpreassembled. It is expected that fully-automated ICC can be employed tolink these building blocks in all possible combinations to generate inprotected form four neolignan natural products and 20 novel neolignananalogs (FIG. 4B). After removing the hydroxyl protecting groups andautomated HPLC purification, all of these natural products and naturalproduct derivatives are expected to be produced in quantities suitablefor biological assays.

The development of a fully automated ICC platform for small moleculesynthesis represents an important step towards increasing the efficiencyand flexibility with which small molecules can be prepared in thelaboratory. While certain types of small molecules (for example, thosepossessing many Csp²-Csp² linkages) are at present more amenable to thisapproach than others, the rapidly expanding scope of the Suzuki-Miyaurareaction, which increasingly includes Csp³ coupling partners (M. R.Netherton, G. C. Fu, Adv. Synth. Catal. 2004, 346, 1525-1532) suggestthat the potential generality of this approach is substantial. Thissynthesis apparatus stands to extend the power of small moleculesynthesis to the non-chemist and ultimately will help shift therate-limiting step in small molecule science from achieving syntheses tounderstanding function. Given that the functional capacity for smallmolecules likely extends far beyond that which is currently understoodor utilized, the developments described herein stand to have widespreadimpacts in both science and medicine.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 211.1.03.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following, which is included merely forpurposes of illustration of certain aspects and embodiments of thepresent invention, and is not intended to limit the invention.

Example 1—General Apparatus Design Principals

One example of an automated small molecule synthesizer is shown in FIG.1D (photograph) and FIG. 5 (schematic). This custom designed apparatuscan execute the fully automated synthesis of eight small moleculessimultaneously. Each synthesis consists of between one and threeiterative coupling sequences, where each coupling sequence can include adeprotection step, a cross-coupling step and a purification step. Theorganization of the apparatus is centered on eight main syringe pumps.Each main syringe pump is dedicated to only one synthesis. These eightmain syringe pumps operate independently to execute iterative couplingsequences in parallel. Resources for each synthesis arecompartmentalized such that each main syringe pump does not access theresources of another main syringe pump, with the following exceptions:all solvents and all product output ports are shared by all of the mainsyringe pumps. Additionally, an auxiliary syringe pump is used as ashared resource for the purification steps. Another auxiliary syringepump is used as a shared resource to handle all aqueous solutions. Thecustom designed software that operates the machine governs how theshared resources are distributed.

Standard Valve.

The valve modules were purchased from J-KEM Scientific (part #Syr-CS4)and are connected to the controlling computer via a RS485 to USBconnection. Each valve module is equipped with four eight-portstream-selecting valves (J-KEM, part #SPDV-CS8). Each valve connects theinput stream, which enters through the center of the valve, to one ofeight possible output streams (ports A thru H). The location of thestandard valves is shown in FIG. 5.

Syringe Pump Valve.

Each syringe pump is fitted with an eight-port stream selecting valve(J-KEM, part #SPDV8) where the input stream enters from the syringeconnected immediately in front of port “E”. Port “E” is partiallyblocked by the syringe and requires a flush-net fitting (IDEX Health andScience, part #F-358) to connect the output stream. The location of thesyringe pump valves is shown in FIG. 5.

Syringe Pumps.

Syringe pumps were purchased from J-KEM Scientific (part #SYR1400-8 forP1-P8, part #SYR-1400PC for P9 and P10) and are connected to thecontrolling computer via a RS485 to USB connection. Each syringe pump isfitted with an eight-port stream-selecting valve (J-KEM, part #SPDV8)and a 10 mL glass syringe equipped with a Teflon plunger (J-KEM, part#SPGS-10000). The syringe pump withdraws and injects at rates from 0.0mL/min to 70.0 mL/min with a step of 0.0029 mL. The location of thesyringe pumps is shown in FIG. 5.

Reaction Tubes.

To minimize cross-contamination and allow the rapid setup of asynthesis, all chemical manipulations are performed in disposablepolypropylene tubes purchased from Luknova, item #FC003012. Thedimensions of the tube are 21 mm×120 mm (ID×length). The bottom of thetube is fitted with a 21 mm diameter×5 mm tall fit. The bottom of thetube is accessed through a male Luer tip, while the top of the tube issealed with an air-tight, threaded cap containing a female Luer port.The tube holds a solvent volume of up to 25 mL. Examples of reactiontubes are shown in FIGS. 6 and 8.

Tubing and Fittings.

All tubing and fittings were purchased from IDEX Health and Science. Thetubing used in the machine is 0.030 inch (ID)× 1/16 inch (OD) Teflon FEPtubing (part #1520 xL). All tubing connections were made with 1/16 inchETFE flangeless ferrules (part #P-200x) and ¼-28 acetal fittings (part#P-202x). Male Luer fittings (part #P-625) and female Luer fittings(part #P-658) are ETFE and PEEK, respectively. Examples of tubes andfittings are shown in FIG. 6.

Example 2—Computer Control of Apparatus

General Programming Design Principals.

All apparatus equipment is controlled by a custom program written inVB.NET using Microsoft Visual Basic 2008 Express Edition. The apparatusis controlled by a single computer running Windows Vista. The apparatusis designed to run an experiment independently on each of the eight mainsyringe pumps. Each main pump can be started and stopped at will withoutaffecting the other pumps. Further, the scripts of simultaneouslyexecuting experiments do not need to be the same nor do they need to besynchronized in order for the experiments to perform successfully. Theprogram is designed to manage distribution of shared resources such ascommon valve equipment and auxiliary syringe pumps as well as bandwidthon the COM ports.

Setting up and modifying the script used to execute an experiment isdesigned to be straightforward and facile. Towards this end, a simplecustom scripting language was developed. The scripting language containsa small collection of commands used to send instructions to a virtualmachine, manage the timing of these operations, and lock/unlock accessto shared resources. The program serves as an interpreter for thiscustom scripting language and maps the virtual machine instructions tothe required apparatus hardware. In this way the same script can be usedon any of the main syringe pumps without modification.

Communication to the Apparatus Equipment.

Commands are sent to the apparatus equipment using the computer's serialports. The RS485 serial ports of the apparatus equipment are connectedto a serial-to-USB converter which is connected to the computer. EachCOM port can address up to 16 pieces of equipment, where a piece ofequipment is defined as a valve or a syringe pump. The syringe pump mayor may not additionally control a solenoid valve. As configured,communication to the apparatus is distributed across four COM ports.Commands can be sent to the equipment no faster than every 20milliseconds. To enforce this delay, the program maintains a queue ofcommands to be sent to the equipment. Commands recognized by theequipment are: move the valve to a specific port, report the currentposition of the valve, move the syringe plunger to a specific positionat a specific rate, turn the solenoid valve on/off, and report if theequipment has finished executing the previous command.

Program Architecture.

Each valve and each syringe pump is represented programmatically as an“equipment object”. Each of these equipment objects is given anidentifier that can be used to map the commands of the scriptinglanguage to the actual hardware represented by the equipment object.Each main pump is assigned a “pump object” that is a container for allof the equipment objects that represent resources that are available tothe main pump. No single pump object contains every equipment object,but every equipment object is contained in at least one pump object.When an experiment is executed on the main pump, the list of scriptedcommands is passed as a text file to the corresponding pump object. Thepump object is responsible for proof-reading, interpreting and executingthe scripted commands and updating the graphic user interface (GUI) asappropriate.

Script Execution.

When the pump object is passed a text file of command lines, the pumpobject interprets each command line into a “command object”. The commandobject contains all of the information that is necessary to execute thecommand. Once a command object is created it is entered into a queuemanaged by the pump object. Through this process the script isproof-read to identify any syntax errors that would prevent the codefrom executing properly. The script is then executing by retrieving anitem from the queue, sending the appropriate commands to the equipmentvia the COM port, periodically checking the status of the machineequipment until the command is complete, and then repeating the processfor the next command in the queue.

Scripting Language.

The scripting language contains the following commands with indicationson their usage:

' (apostrophe) Indicates everything following the apostrophe is acomment and should not be interpreted. [text] (brackets) The bracketsrepresent a placeholder value recognized by the identifier text thatwill be replaced at runtime with the define command. define text=valueAt runtime replaces all brackets (placeholders) containing text with thevalue of value. This a useful strategy for writing flexible scripts.pause n Halts execution of the script for n seconds. valve nL Movesvalve n to port position L (A-H). valve xon valve xon opens the solenoidvalve associated valve xoff with the main syringe pump. valve xoffcloses this solenoid valve. pump n in=L out=M Fills the syringe with nmL drawn from port ratein=x rateout=y position L (A-H) at a rate of xmL/min. and inject n mL out through port position M(A-H) at y mL/min.The port position refers to the valve that is directly connected to thesyringe. Omitting the in=portion instructs the pump to only dispense nmL as per above. Omitting the out=portion instructs the pump to onlyfill n mL as per above. Use rate=x to set both the rate of withdrawaland the rate of injection to x mL/min. pump out=M rate=x Injects theentire contents of the syringe out through port position M(A-H) at arate of x mL/min. log “comment text” Writes a time-stamped entry to thelog book containing the user-defined text (comment text). lock n Claimsvalve n for the exclusive use by the main pump. If valve n is busy orhas been locked by a different pump, execution of the script is halteduntil the valve becomes available. Auxiliary pumps can also be lockedusing this command. unlock n Releases valve n from the exclusive use bythe main pump. All lock commands should be eventually followed by anunlock command. Auxiliary pumps can also be released using this command.sub sub_name The sub and end sub commands mark the end sub beginning andend, respectively, of command lines that will be interpreted as a subroutine with the identifier sub_name. run sub_name Runs the sub routineidentified as sub_name. This sub routine should have been previouslydefined using the sub and end sub commands. The command following therun command is not executed until all of the commands in the sub routinehave completed (as opposed to the background command.) backgroundsub_name Runs the sub routine identified as sub_name. This command issimilar to the run command except that as soon as the sub routine beginsto execute, the command following the background command executes aswell. Therefore, the sub routine is handled as a background processallowing multiple actions to be performed at once. wait sub_name Haltsexecution of the script until the sub routine identified as sub_name(which was previously executed using the background command) finishesits execution. This command allows background processes to besynchronized with the main script.

Example 3—Chemical Synthesis

General Procedure.

All chemical manipulations were performed in polypropylene tubespurchased from Luknova, item #FC003012. The dimensions of the tube are21 mm×120 mm (ID×length). The bottom of the tube is fitted with a 21 mmdiameter×5 mm tall frit. The bottom of the tube is accessed through amale Luer tip, while the top of the tube is sealed with an air-tight,threaded cap containing a female Luer port. The tube holds a solventvolume of up to 25 mL.

Deprotection Tubes.

To enable automation, a novel MIDA boronate deprotection method wasdeveloped using Amberlyst A26(OH) resin. Amberlyst A26(OH) was purchasedfrom Sigma-Aldrich and was stored under N₂ atm. at 4° C. AmberlystA26(OH) (suspension volume of 20 mL) was twice washed with MeCN (50 mL)with vigorous agitation for 60 seconds in each wash. The residualsolvent was evaporated under a fast stream of air for 5 minutes untilthe resin was light beige in color and was free-flowing. To eachpolypropylene tube was added the Amberlyst resin (2.0 g resin for every1.0 mmol of MIDA boronate to be deprotected) and, optionally, the MIDAboronate starting material. The tube was capped and then placed on themachine where the bottom Luer tip connected to the deprotection tableand the top Luer port is covered with aluminum foil.

Drying and Degassing Tubes.

A polypropylene tube was charged with Celite®, activated molecularsieves (4 Å, 8-12 mesh) and K₂CO₃. The amounts of these reagents areproportional to the amount of Amberlyst A26 resin used in thedeprotection step prior to drying/degassing, as indicated below. Ontothe bed of solids was placed a plastic plunger, cut from the plunger ofa 5 mL polypropylene syringe (Fisher #14-817-28, Norm-Ject). The plungerprevents the solids from lifting during the degassing step. The tube wascapped and then placed on the machine where the bottom Luer tip connectsto the degassing table and the top Luer port is connected to the gasmanifold.

Amberlyst A26 (previous step) Celite ® Mol. sieves K₂CO₃ 2.0 g 200 mg2.0 g 2.0 g 1.0 g 100 mg 1.0 g 1.0 g 0.5 g  50 mg 0.5 g 0.5 g

Reaction Tubes.

To assist in the transfer of small amounts of Pd(OAc)₂ and S-Phos, thesereagents were adsorbed onto Cs₂CO₃ as follows. To a 40 mL glass vial wasadded Pd(OAc)₂ (22 mg) and Cs₂CO₃ (2.723 g). To the vial was added THF(10 mL), and the suspension was concentrated in vacuo to afford a paleamber powder, the Pd-mixture. To a 40 mL glass vial was added S-Phos (76mg) and Cs2CO3 (2.667 g). To the vial was added THF (10 mL), and thesuspension was concentrated in vacuo to afford a white powder, theSPhos-mixture.

To a polypropylene tube was added a stir bar (Big Science Inc.,SBM-1508-REH), the halide (0.333 mmol), the Pd-mixture (488 mg, 5% Pd)and the SPhos-mixture (488 mg, 10% S-Phos). For aqueous couplings, tothe tube was added a KOH pellet (75 mg, 1.7 mmol). The tube was cappedwith a modified cap (see detail) and was placed in a heating block. Thebottom of the tube is connected the reaction table, the top of the tubeis vented to the gas manifold, and the second top input is connected tothe reaction table for addition of the boronic acid. Building on thepreviously published reports of “slow-release” cross-coupling, theboronic acid was added slowly via syringe pump to minimize in situdecomposition and thereby maximize yields.

Automation.

Each cross-coupling in the automated sequence was performed according tothe following, fully automated script:

Deprotection

-   -   1) Add THF (5 mL) to deprotection tube    -   2) Agitate the mixture via gas bubbling for 60 minutes    -   3) Add AcOH in THF (4.0 M, 5.0 mmol per 1.0 g of Amberlyst        resin)    -   4) Agitate the mixture via gas bubbling for 15 minutes    -   5) Transfer the solution to the drying tube, washing the resin        with THF (5×1.0 mL)    -   6) Sparge the mixture with Ar gas for 15 minutes    -   7) While sparging the reaction tube with Ar gas for 15 min.,        agitate the THF mixture via gas bubbling every 2 minutes

Cross-Coupling

-   -   8) Add THF (3 mL) to the reaction tube and allow the mixture to        stir for 10 min.    -   9) Transfer the boronic acid solution from the drying tube to        the reaction tube over 120 min., washing the solids with THF        (3×1.0 mL)    -   10) Stir the reaction mixture at 150 rpm for 22 hours.

Purification

-   -   11) Add hexanes (12 mL) to the ppt. chamber, then add a portion        of the reaction solution (3 mL) to the ppt. chamber.    -   12) Withdraw the solution in the ppt. chamber through the SiO₂        plug and send to waste    -   13) Repeat steps 11 and 12 until all of the reaction solution        has been transferred    -   14) Add Et₂O w/MeOH (1.5% v/v) (7.5 mL) to the ppt. chamber,        withdraw the solution through the SiO₂ plug, and send to waste.        Repeat an additional four times.    -   15) Add Et₂O (7.5 mL) to the ppt. chamber, withdraw the solution        through the SiO₂ plug, and send to waste. Repeat an additional        two times.    -   16) Flow Ar gas through the SiO₂ plug for three minutes to        evaporate residual solvent.    -   17) Add THF (6.8 mL) to the ppt. chamber    -   18) Withdraw the THF solution through the SiO₂ plug and then        inject the solution back into the ppt. chamber. Repeat an        additional two times.    -   19) Withdraw the THF solution through the SiO₂ plug and inject        the solution into the deprotection tube used in the next        reaction. The next reaction begins at step 1.

Direct Release/Aqueous Coupling Modification (Typically Performed as theLast Step of an Automated Synthesis).

This sequence begins after step 19 from the general automation script(above).

-   -   1) Sparge the THF solution derived from the purification of the        previous cross-coupling with Ar gas for 15 minutes.    -   2) Transfer the THF solution to the reaction tube in one        portion.    -   3) Stir the mixture for 5 minutes.    -   4) Add degassed H₂O (2 mL) to the reaction tube    -   5) Stir the reaction mixture at room temperature for 12 hours.    -   6) Add aq. NH₄Cl (2.5 mL), mix for 5 minutes, then withdraw the        entirety of the mixture and transfer it to the product test        tube.

β-Parinaric Acid.

All steps were performed according to the general procedure. The machinewas equipped with reagent tubes as follows.

Step 1. The machine was equipped with a deprotection tube charged withAmberlyst resin (2.0 g) and MIDA boronate 1 (211 mg, 1.0 mmol); a dryingtube charged with molecular sieves (2.0 g), K₂CO₃ (2.0 g) and Celite®(0.2 g); and a reaction tube charged with the Pd-mixture (488 mg), theSPhos-mixture (488 mg) and boronate 2 (103 mg, 0.333 mmol).

Step 2. The machine was equipped with a deprotection tube charged withAmberlyst resin (1.0 g); a drying tube charged with molecular sieves(1.0 g), K₂CO₃ (1.0 g) and Celite® (0.1 g); and a reaction tube chargedwith the Pd-mixture (244 mg), the SPhos-mixture (244 mg), and boronate 2(34 mg, 0.11 mmol).

Step 3. The machine was equipped with a deprotection tube (empty, butused for sparging the MIDA boronate solution) and a reaction tubecharged with the Pd-mixture (60 mg), the SPhos-mixture (60 mg), KOH (75mg, 1.7 mmol) and halide 3 (11 mg, 0.037 mmol).

Automation.

The synthesis was performed in a fully automated fashion with nooperator intervention. Step 1 and step 2 were performed following thestandard script, and step 3 was performed following the directrelease/aqueous coupling modification of the standard script. Theaqueous mixture that was outputted from the machine was manuallypurified as follows: The mixture was transferred to a 60 mL separatoryfunnel and was diluted with sat. aq. NH₄Cl (10 mL). The mixture wasextracted twice with Et₂O (20 mL) and the combined organics were washedwith brine (20 mL); dried over MgSO₄; filtered, then concentrated invacuo. The yellow residue was purified via SiO₂ chromatography to affordβ-parinaric acid as a white solid (yield not yet determined). The ¹H-NMR(CDCl₃) spectrum of the synthesized product was fully consistent withthe literature data (Lee, S. J.; Gray, K. C.; Paek, J. S.; Burke, M. D.J. Am. Chem. Soc., 2008, 130, 466-468).

To characterize the efficiency of each step and characterize allintermediates, Step 1 was repeated and the MIDA boronate solutiongenerated in line 19 of the standard script was diverted into a testtube and then concentrated to afford pure intermediate 4 as a colorlesssolid (40 mg, 52%).

¹H-NMR (500 MHz, acetone-d₆): δ 6.53 (dd, J=17.0, 10 Hz, 1H), 6.11 (dd,J=15.5, 10.0 Hz, 1H), 5.82 (dt, J=15.5, 6.5 Hz, 1H), 5.54 (d, J=17.5 Hz,1H), 4.20 (d, J=17.0 Hz, 2H), 4.01 (d, J=17.0 Hz, 2H), 2.98 (s, 3H),2.10 (quint, J=7.5 Hz, 2H), 0.99 (t, J=7.5 Hz, 3H). ¹³C-NMR (125 MHz,acetone-d₆): δ 196.1, 143.7, 137.8, 132.6, 62.2, 47.2, 26.1, 13.7.

Step 1 and step 2 were repeated and the MIDA boronate solution generatedduring the second coupling (line 19 of the standard script) was divertedinto a test tube and then concentrated to afford pure intermediate 5 asa colorless solid (22 mg, 76%).

¹H-NMR (500 MHz, acetone-d₆): δ 6.58 (dd, J=18.0, 10.5 Hz, 1H), 6.28(dd, J=15.0, 10.0 Hz, 1H), 6.20 (dd, J=15.0, 10.0 Hz, 1H), 6.11 (ddt,J=15.5, 10.5, 1.5 Hz, 1H), 5.82 (dt, J=15.0, 6.5 Hz, 1H), 5.64 (d,J=17.5 Hz, 1H), 4.21 (d, J=17.0 Hz, 2H), 4.03 (d, J=17.0 Hz, 2H), 2.99(s, 3H), 2.12 (quint, J=7.5 Hz, 2H), 0.99 (t, J=7.5 Hz, 3H). ¹³C-NMR(125 MHz, acetone-d₆): δ 169.0, 143.5, 138.3, 134.8, 133.5, 130.3, 62.2,47.3, 26.4, 13.8.

All-Trans-Retinal.

All steps were performed according to the general procedure. The machinewas equipped with reagent tubes as follows.

Step 1. The machine was equipped with a deprotection tube charged withAmberlyst resin (1.0 g) and MIDA boronate 6 (173 mg, 0.500 mmol); adrying tube charged with molecular sieves (1.0 g), K₂CO₃ (1.0 g) andCelite® (0.1 g); and a reaction tube charged with the Pd-mixture (244mg), the SPhos-mixture (244 mg) and boronate 2 (52 mg, 0.111 mmol).

Step 2. The machine was equipped with a deprotection tube charged withAmberlyst resin (0.5 g); a drying tube charged with molecular sieves(0.5 g), K₂CO₃ (0.5 g) and Celite® (50 mg); and a reaction tube chargedwith the Pd-mixture (82 mg), the SPhos-mixture (82 mg). A separatepolypropylene tube was charged with a solution of halide 7 (0.056 mmol)in degassed THF (3 mL).

Automation.

The synthesis was performed in a fully automated fashion with nooperator intervention. Step 1 was performed following the standardscript. Step 2 was performed following the standard directrelease/aqueous coupling modification script with the additionalmodification that the THF used in line 8 of the standard script was theentirety of the solution containing halide 7. Further, the script forstep 2 was stopped after line 10 and the reaction solution was outputtedto a test tube. The product was manually purified as follows: Thereaction solution was concentrated in vacuo and the solid yellow residuewas purified by SiO₂ chromatography using an Isco-Teledyne CombiFlashsystem to afford all-trans-retinal as a yellow solid (3.3 mg, 20%). The¹H-NMR (CDCl₃) spectrum of the product was fully consistent with theliterature data (Lee, S. J.; Gray, K. C.; Paek, J. S.; Burke, M. D. J.Am. Chem. Soc., 2008, 130, 466-468).

Example 4—Generality of Purification Platform

To establish the generality of the novel purification platform, itscapacity to purify a series of MIDA boronates representing a diverserange of structures, including aryl, heteroaryl, alkynyl, alkenyl, andalkyl derivatives, was tested. Briefly, mock crude reaction mixtureswere prepared by mixing each MIDA boronate (1 equiv.) (Table 1) withtolyl-boronic acid (1 equiv.) and a palladium catalyst (0.1 equiv) inTHF. Each of these mixtures was then subjected to fully-automatedpurification via the hybrid precipitation/catch-and-release platformdescribed in detail herein. At the end of this process, all of theseMIDA boronates were obtained in >90% purity as judged by ¹H NMR (seeFIGS. 11-26), and the yields of recovered MIDA boronates were good tooutstanding (Table 1).

TABLE 1 Purification from mock crude reaction mixtures

Boronate % Recovery

69

53

92

92

92

83

81

78

76

86

87

90

68

94

86

65

Details of the procedure are as follows.

Pre-Activation of the Catalyst Solution:

Palladium(II) acetate (0.001875 mmol, 2.5 mol %) and SPhos (0.00375mmol, 5 mol %) per purification to be run were combined in an 8 mLscintillation vial equipped with a PTFE-coated magnetic stir bar andplaced under an argon atmosphere. THF was added to generate a 0.01 Mcatalyst stock solution (with respect to palladium(II) acetate), and itwas stirred vigorously for 30 min at room temperature to generate anorange, yellow, or clear solution. After this activation process, 1 mLcatalyst stock solution was added to the solution in the polypropylenecartridge containing the simulated reaction mixture.

Preparation and Installation of Simulated Reaction Chamber:

A new fritted 12 g Luknova polypropylene cartridge was charged with MIDAboronate (0.075 mmol, 1 eq), 4-methylbenzene boronic acid (0.075 mmol, 1eq), and THF (10 mL). After addition of the pre-activated catalystsolution, the cartridge was installed into the Luer fittings in thereaction block of the automated synthesizer. Once all cartridges were inplace, the automated purification routine was run using the computerinterface. The samples were collected as THF solutions into tared 40 mLscintillation vials.

Concentration, Azeotropic Drying, and Analysis of Recovered Materialsfrom Purifications:

The THF solutions were concentrated under reduced pressure on a rotaryevaporator, then the residue was azeotroped with dichloromethane (3×5mL) to remove residual solvents. These residues were then placed undervacuum for 12-36 hours, after which yield and purity were determined bycomparison of ¹H NMR in acetone-d6 with a standard sample of the desiredMIDA boronate and with a sample taken of a simulated reaction mixture.

Automated Purification Detailed Protocol

-   -   1) In the background, auxiliary pump aspirates 6 mL hexanes and        delivers it to the bottom of the precipitation chamber, through        the silica gel column. This process is repeated once for a total        of 12 mL hexanes.    -   2) Primary pump aspirates 9 mL of reaction mixture from reaction        chamber bottom and returns 6 mL, through bottom, to ensure no        more than 3 mL will be delivered to the precipitation chamber.    -   3) Primary pump delivers 3 mL of reaction mixture to top of        precipitation chamber containing 12 mL hexanes. This induces        MIDA boronate precipitation from the THF solution. Primary pump        then delivers two 10-mL plugs of dry nitrogen to bottom of        precipitation chamber (bypassing the silica gel column) to        dislodge stir bar.    -   4) Suspension in precipitation chamber is aspirated from bottom        and through silica gel column by auxiliary pump. Eluent is sent        to waste.    -   5) Steps 1-4 repeat three additional times to send all of        reaction mixture to precipitation chamber.    -   6) Primary pump aspirates 1.5 mL THF and delivers it to the top        of reaction chamber as a rinse. Steps 1-3 are repeated.    -   7) Primary pump aspirates 1.5 mL THF and delivers it to top of        reaction chamber as a rinse. Steps 2-3 are repeated.    -   8) Step 4 is repeated.    -   9) Steps 1-4 are repeated.    -   10) Step 4 is repeated.    -   11) Primary pump aspirates 6.5 mL 1.5% (v/v) MeOH in Et₂O and        delivers it to top of precipitation chamber. This process is        repeated once for a total delivery of 13 mL solvent.    -   12) Primary pump delivers two 10-mL plugs of dry nitrogen to the        bottom of the precipitation chamber (bypassing the silica gel        column) to dislodge stir bar.    -   13) Step 4 is repeated.    -   14) Steps 11-13 are repeated. Step 4 is repeated again.    -   15) Steps 11-13 are repeated twice with Et₂O instead of 1.5%        (v/v) MeOH in Et₂O. Step 4 is repeated twice more to dry out        silica gel column.    -   16) Auxiliary pump is rinsed with 2×1 mL THF to wash away any        residual MeOH. Wash THF is sent to waste.    -   17) Auxiliary pump aspirates 6 mL THF and delivers slowly to        bottom of precipitation chamber through silica gel column. This        process is repeated once for a total of 12 mL THF.    -   18) Primary pump aspirates 5 mL dry nitrogen and delivers it to        bottom of precipitation chamber (bypassing the silica gel        column) to agitate the suspension, thus promoting mixing MIDA        boronate dissolution. This process is done 40 times.    -   19) THF solution of MIDA boronate is aspirated by primary pump        out of the bottom of the precipitation chamber (bypassing the        silica gel column). Solution is delivered to the collection        tube. This aspiration/delivery is repeated an additional 5 times        to ensure full transfer.    -   20) Auxiliary pump pushes residual THF in silica gel column into        bottom of precipitation chamber as a rinse.    -   21) Primary pump aspirates 5 mL dry nitrogen and delivers it to        bottom of precipitation chamber (bypassing the silica gel        column) to agitate the suspension, thus promoting mixing MIDA        boronate dissolution. This process is done 5 times.    -   22) THF rinse is aspirated by primary pump out of bottom of the        precipitation chamber (bypassing the silica gel column).        Solution is delivered to the collection tube.

The results from this study of a wide range of structurally diverse MIDAboronates demonstrates that the hybrid precipitation/catch-and-releasepurification strategy is remarkably general.

Example 5—Aqueous Deprotection Module

Automated aqueous deprotection of phenyl MIDA boronate, trienyl MIDAboronate, and butenyl MIDA boronate was performed using the aqueousdeprotection strategy and module described above.

Automated aqueous deprotection of phenyl MIDA boronate afforded phenylboronic acid. Subsequent automated cross-coupling with the vinyl iodidebifunctional building block afforded the desired coupled product in 77%yield after manual purification. See FIG. 28A.

Automated aqueous deprotection of the trienyl MIDA boronate afforded thetrienyl boronic acid. Subsequent automated cross-coupling with the vinyliodide bifunctional building block afforded the desired coupled productin 66% yield after manual purification. See FIG. 28B.

Automated aqueous deprotection of the butenyl MIDA boronate afforded thebutenyl boronic acid. Subsequent automated cross-coupling with anisomeric mixture of the dienyl vinyl iodide bifunctional building blockafforded a 75% yield of the expected isomeric trienyl coupled productsin a 75% yield after automated purification. See FIG. 28C.

Example 6—Fully Automated Synthesis of All-trans-Retinal Using AqueousDeprotection Module

The First Deprotection Tube was Prepared as Follows:

To a new, fitted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was added trienyl MIDA boronate (345.2 mg, 1 mmol, 9 equivs).To this was added sodium hydroxide (120.0 mg, 3 mmol, 27 equivs). Thecartridge was capped with its female luer-port screw cap. To this Luerport was attached a 5-mL polypropylene syringe barrel (Henke-Sass, WolfGmbH, Tuttlingen, Germany, 78532, Part #A5) from which the plunger hadbeen removed. This first deprotection tube was wrapped with aluminumfoil.

The Second Deprotection Tube was Prepared as Follows:

To a new, fritted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was added sodium hydroxide (40.0 mg, 1 mmol, 9 equivs).Sodium hydroxide pellets were shaved down to the correct mass with aclean razor blade and massed quickly to minimize adsorption ofatmospheric moisture. The cartridge was capped with its female luer-portscrew cap. To this Luer port was attached a 5-mL polypropylene syringebarrel (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part #A5)from which the plunger had been removed. This second deprotection tubewas wrapped with aluminum foil.

The First and Second Predrying Tubes were Prepared as Follows:

To a new, fitted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was added Celite® 545 filter aid (800 mg, not acid-washed,Acros Organics, Product #349670025, Lot #A0287832). To this was addedanhydrous magnesium sulfate (2.1 g, ReagentPlus®, ≧99.5%, Sigma-Aldrich,Product #M7506, Lot #080MO246V). These two solids were mixed with aspatula until visibly homogenous. On top of the solid mixture was placeda 5-mL polypropylene syringe plunger (Henke-Sass, Wolf GmbH, Tuttlingen,Germany, 78532, Part #A5), manually cut to approximately 6.5 cm inlength. The cartridge was capped with its female luer-port screw cap.The Luer port was covered tightly with a small square (approximately 1cm×1 cm) of aluminum foil. Each predrying tube was wrapped with aluminumfoil.

The First and Second Drying Tubes were Prepared as Follows:

To a new, fritted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was added Celite® 545 filter aid (300 mg, not acid-washed,Acros Organics, Product #349670025, Lot #A0287832). To this was addedactivated molecular sieves (3.6 g, 4 Å, −325 mesh, Sigma-Aldrich,Product #688363, Lot #MKBF4010V). Molecular sieves were activated at300° C., ambient pressure, 24 h, and cooled/stored in a vacuumdesiccator under dry argon over Drierite. These two solids were notmixed. On top of the layered solids was placed a 5-mL polypropylenesyringe plunger (Henke-Sass, Wolf GmbH, Tuttlingen, Germany, 78532, Part#A5), manually cut to approximately 5.5 cm in length. The cartridge wascapped with its female luer-port screw cap. Each drying tube was wrappedwith aluminum foil.

The First and Second Deoxygenating/Concentrating Tubes were Prepared asFollows:

A new, fritted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was capped with its femal luer-port screw cap. Eachdeoxygenating/concentrating tube was wrapped with aluminum foil.

The First Reaction Tube was Prepared as Follows:

To a new, fritted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) was added a 4-g frit (Luknova, Mansfield, Mass., 02048, Part#FC003004). This frit was secured, concentrically, to the 12-g cartridgefrit with 26 G Chromel A wire, pierced through the 12-g frit. To thisreaction tube was added, in order, anhydrous potassium phosphate (1.39g, 3 mmol+750 mg, 27 equivs+750 mg, 97%, Alfa Aesar, Product #L15168,Lot #L02U015), palladium (II) acetate (1.9 mg, 0.0083 mmol, 2.5 mol %,≧99.9%, Sigma-Aldrich, Product #520764, Lot #1000824996),2-dicyclohexylphosphino-2′,6′-dimethyoxy-1,1′-biphenyl (Sphos, 6.8 mg,0.017 mmol, 5 mol %, 98%, Strem Chemicals, Product #15-1143, Lot#18526300), vinyl iodide MIDA boronate (103.0 mg, 0.33 mmol, 3 equivs),and a PTFE-coated rare earth magnetic stir bar. Potassium phosphate wasfreshly ground in a 100° C. mortar and pestle. The cartridge was cappedwith its customized female luer-port screw cap. The customized capconsists of a standard female luer-port cap with a bent (byapproximately 45°), 1.5 inch, 18 G, disposable needle installed throughthe cap and a small ball of Kimwipe inserted into the Luer port. It isimportant remove the cored-out polypropylene plug from the inside of theneedle after installation. The cap was topped with a fitted 4-gcartridge (Luknova, Mansfield, Mass., 02048, Part #FC003004).

The Precipitation Tube was Prepared as Follows:

To a new, fritted 12-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003012) equipped with a PTFE-coated magnetic stir bar was addedCelite® 545 filter aid (150 mg, not acid-washed, Acros Organics, Product#349670025, Lot #A0287832) and 3-aminopropyl functionalized silica gel(250 mg, 40-63 μm, approximately 1 mmol/g NH₂, Sigma-Aldrich, Product#364258, Lot #79096HM). The cartridge was capped with its femaleLuer-port screw cap. To the cartridge was added hexanes (5 mL, reagentgrade) and the resulting suspension was swirled vigorously to mix thesolids. The mixed suspension was allowed to settle for approximately 5seconds and then the solvent was drained by forcing a plug of ambientair through the top of the cartridge by syringe. This process firmlyembeds the stir bar in the solids to prevent stirring before theprecipitation tube is utilized. This precipitation tube was wrapped withaluminum foil.

The Silica Gel Chromatography Column was Prepared as Follows:

A silica gel chromatography column was freshly prepared from custom PTFEfittings using unfunctionalized silica gel. The cartridge was modeledafter a 4-g cartridge (Luknova, Mansfield, Mass., 02048, Part#FC003004), but was made of PTFE instead of polypropylene. To a clean,fritted column was added silica gel. This was done by vacuum aspirationthrough the bottom male Luer tip fitting. This process ensured tight,even packing of the silica gel plug. Excess silica gel was removedmanually with a spatula and a 4-g frit (Luknova, Mansfield, Mass.,02048, Part #FC003004) was placed on top of the silica plug. Thisdoubly-fritted cartridge was capped with its female luer-port screw cap,using PTFE tape to ensure a tight seal.

The Second Reaction Vessel was Prepared as Follows:

To a non-flame-dried 7-mL glass vial equipped with a PTFE-coatedmagnetic stir bar was added palladium (II) acetate (1.2 mg, 0.0056 mmol,5 mol %, ≧99.9%, Sigma-Aldrich, Product #520764, Lot #1000824996),2-dicyclohexylphosphino-2′,6′-dimethyoxy-1,1′-biphenyl (Sphos, 4.6 mg,0.011 mmol, 10 mol %, 98%, Strem Chemicals, Product #15-1143, Lot#18526300), and anhydrous potassium phosphate (212 mg, 1 mmol, 9 equivs,97%, Alfa Aesar, Product #L15168, Lot #L02U015). Potassium phosphate wasfreshly ground in a 100° C. mortar and pestle. This vial was sealed witha PTFE-lined septum screw cap. Through the septum was added a 1.5 inch,20 G, disposable needle connected to a dry argon gas line. Then, throughthe septum was added a 1.5 inch, 20 G, disposable needle to act as avent. The reaction vial was then flushed with dry argon forapproximately 7 min. The vent needle and then the argon needle wereremoved from the septum.

The Tubes, Vessels and Columns Described Above were Used as Follows:

See FIG. 29 for reaction scheme. Both deprotection tubes (wrapped withaluminum foil) were securely installed on the machine. Tubes wereinstalled by placing the tube's male Luer tip into the machine'sappropriate female Luer port and were secured with a firm downward forceand slight (less that one quarter turn) clockwise rotation. Bothpredrying tubes (wrapped and topped with aluminum foil) were securelyinstalled on the machine. Both drying tubes (wrapped in aluminum foil)were securely installed on the machine. Each drying tube was connectedto the inert gas manifold by attaching a patch line to the machine's gasmanifold and the tube's top Luer port. Patch lines are approximately12-inch lengths of tubing with male luer-tip fittings on both ends. Bothdeoxygenating/concentrating tubes (wrapped in aluminum foil) weresecurely installed on the machine. Each deoxygenating/concentrating tubewas connected to the inert gas manifold by attaching a vented patch lineto the machine's gas manifold and the tube's top Luer port. Vented patchlines are approximately 12-inch lengths of tubing with a male luer-tipfitting on the machine-end and a Y-connector (one port connected to theline, one port connected to a male luer-tip fitting, and one port leftopen) on the tube-end. The first reaction tube was securely installed onthe machine (in a heating block preheated to 40° C.) and connected tothe inert gas manifold by attaching the reaction vent line to the tube'stop Luer port. The reaction tube was then covered with aluminum foil andset to stirring at 600 rpm. The silica gel column was securely installedon the machine and connected to the purification module by attaching theeluent line to the column's top Luer port. The precipitation tube(wrapped in aluminum foil) was securely installed on the machine andconnected to the purification module by placing the eluent line (fixedwith a 1.5 inch, 18 G, disposable needle) through the tube's top Luerport).

The experiment's pre-assembled code was then loaded and executed tobegin the automated sequence. The first aqueous MIDA boronatedeprotection commenced immediately. After running the first deprotection(rt, 10 min), the machine quenched and worked up the resulting boronicacid solution and then dried, deoxygenated, and concentrated it. Themachine then ran the first, slow addition, cross-coupling reaction (40°C., 8 h total) and purified the resulting coupled product. The machinethen ran the second aqueous MIDA boronate deprotection (rt, 10 min) andsubsequently quenched, worked up, dried, deoxygenated, and concentratedthe resulting boronic acid solution.

Approximately 5 minutes before the second cross-coupling began, thesecond reaction vessel was placed in an aluminum block (at roomtemperature) on a stir plate. An inert gas vent line (fixed with a 1.5inch, 20 G, disposable needle) was connected, through the septum. Thereaction tube was then covered with aluminum foil and set to stirring at600 rpm. Separately, into a non-flame-dried 1.5-mL glass vial was addedthe aldehyde (16.6 mg, 0.11 mmol, 1 equiv). The vial was sealed with aseptum screw cap and to this was added 100 μL deoxygenated dry THF froma 100 μL, gas tight, fixed needle, glass syringe. The vial was manuallygently agitated to dissolve the aldehyde and then was added to thereaction vial with the same syringe. The remaining residual aldehyde wasquantitatively transferred to the reaction vial with 2×50 μL ofdeoxygenated dry THF using the same syringe. As the machineautomatically deoxygenated the reaction addition line (fixed with a 1.5inch, 22 G, disposable needle), it was connected to the reaction vessel,through the septum. The machine then ran the second, fast addition,cross-coupling reaction (rt, 3 h).

At the end of 3 hours, the reaction vial was removed from the machineand the crude reaction mixture was filtered through a 1-cm pad ofCelite® packed in a pipette. The reaction vial was washed with 3×2 mLdry THF and these washings were filtered through the Celite® pad. Thepad was then washed with 3×2 mL dry THF. The resulting clear dark yellowfiltrate was concentrated in vacuo (rt, 80 Torr), azeotroped with 3×5 mLdichloromethane (rt, 80 Torr), and residual solvent was removed on highvacuum (30 min, 200 mTorr) to afford a dark yellow/orange sticky solid.This crude product was manually purified by silica gel flashchromatography to afford a mixture of all-trans-retinal:13-cis-retinalin a ratio of 1:0.55 in a combined total 30% yield.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The appended claims are notintended to claim all such embodiments and variations, and the fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

We claim:
 1. A method of purifying an N-methyliminodiacetic acid (MIDA)boronate from a solution, comprising the steps of diluting the solutionwith hexane, thereby selectively precipitating the MIDA boronate; andisolating the precipitated MIDA boronate, wherein the solutioncomprising the MIDA boronate is a THF solution.
 2. The method of claim1, wherein the isolating comprises filtering the precipitated MIDAboronate.
 3. The method of claim 1, wherein the solution comprising theMIDA boronate is added dropwise to the hexane.
 4. The method of claim 1,wherein the volume of hexane is between about two times and about fourtimes the volume of the solution.
 5. The method of claim 1, wherein thevolume of hexane is about three times the volume of the solution.
 6. Themethod of claim 1, wherein the MIDA boronate is represented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of hydrogen and an organic group.7. The method of claim 6, wherein R²¹, R²², R²³ and R²⁴ are hydrogen. 8.The method of claim 1, wherein the method is performed in thepurification module of an automated small molecule synthesizer, saidautomated small molecule synthesizer comprising: (a) a deprotectionmodule, in fluid communication with (b) a drying and degassing module,in fluid communication with (c) a reaction module, in fluidcommunication with (d) a purification module; (e) at least one pumpwhich can move liquid from one module to another; and (f) a computerequipped with software; wherein all of the modules are under control ofthe computer.
 9. A method of purifying an N-methyliminodiacetic acid(MIDA) boronate from a solution, comprising the steps of passing thesolution through a silica plug; passing a first liquid through thesilica plug; and passing a second liquid through the silica plug,thereby eluting the MIDA boronate in the second liquid, wherein thefirst liquid comprises diethyl ether or the polarity of the first liquidis less than or equal to about the polarity of a mixture of 98.5:1.5(v/v) Et₂O:MeOH; the polarity of the second liquid is greater than orequal to about the polarity of tetrahydrofuran (THF); and the method isperformed in the purification module of an automated small moleculesynthesizer, said automated small molecule synthesizer comprising: (a) adeprotection module, in fluid communication with (b) a drying anddegassing module, in fluid communication with (c) a reaction module, influid communication with (d) a purification module; (e) at least onepump which can move liquid from one module to another; and (f) acomputer equipped with software, wherein all of the modules are undercontrol of the computer.
 10. The method of claim 9, wherein the firstliquid comprises diethyl ether.
 11. The method of claim 9, wherein thefirst liquid is diethyl ether.
 12. The method of claim 9, wherein thefirst liquid is a mixture of diethyl ether and methanol.
 13. The methodof claim 9, wherein first liquid is a mixture of diethyl ether andmethanol; and the ratio of diethyl ether to methanol is 98.5:1.5 (v/v).14. The method of claim 9, wherein the second liquid is THF, MeCN, ethylacetate or acetone.
 15. The method of claim 9, wherein the second liquidis THF.
 16. The method of claim 9, wherein the MIDA boronate isrepresented by

R¹⁰ represents an organic group; B represents boron having sp³hybridization; R²⁰ is methyl; and R²¹, R²², R²³ and R²⁴ independentlyare selected from the group consisting of hydrogen and an organic group.17. The method of claim 16, wherein R²¹, R²², R²³ and R²⁴ are hydrogen.18. A method of purifying an N-methyliminodiacetic acid (MIDA) boronatefrom a solution, comprising the steps of diluting the solution withhexane, thereby selectively precipitating the MIDA boronate; passing thediluted solution through a silica plug, thereby depositing theprecipitated MIDA boronate on the silica plug; passing a first liquidthrough the silica plug; and passing a second liquid through the silicaplug, thereby eluting the MIDA boronate in the second liquid, whereinthe first liquid comprises diethyl ether or the polarity of the firstliquid is less than or equal to about the polarity of a mixture of98.5:1.5 (v/v) Et₂O:MeOH; the polarity of the second liquid is greaterthan or equal to about the polarity of tetrahydrofuran (THF); and themethod is performed in the purification module of an automated smallmolecule synthesizer, said automated small molecule synthesizercomprising: (a) a deprotection module, in fluid communication with (b) adrying and degassing module, in fluid communication with (c) a reactionmodule, in fluid communication with (d) a purification module; (e) atleast one pump which can move liquid from one module to another; and (f)a computer equipped with software, wherein all of the modules are undercontrol of the computer.
 19. A method of deprotecting anN-methyliminodiacetic acid (MIDA) boronate, comprising the step ofcontacting a solution comprising the MIDA boronate and a solvent with asolid-supported ammonium hydroxide reagent, thereby forming a boronicacid and a MIDA, wherein the method is performed in the deprotectionmodule of an automated small molecule synthesizer, said automated smallmolecule synthesizer comprising: (a) a deprotection module comprisingthe solid-supported ammonium hydroxide reagent, in fluid communicationwith (b) a drying and degassing module, in fluid communication with (c)a reaction module, in fluid communication with (d) a purificationmodule; (e) at least one pump which can move liquid from one module toanother; and (f) a computer equipped with software, wherein all of themodules are under control of the computer.
 20. A method of deprotectingan N-methyliminodiacetic acid (MIDA) boronate, comprising the steps ofcontacting a solution comprising the MIDA boronate and a solvent with anaqueous solution of NaOH, thereby forming a boronic acid and free MIDAligand; adding diethyl ether, thereby generating a biphasic mixturecomprising an organic phase comprising the deprotected MIDA boronate andan aqueous phase; and isolating the organic phase comprising the boronicacid and free MIDA ligand; and contacting the organic phase with one ormore drying agents selected from the group consisting of magnesiumsulfate, diatomaceous earth, and molecular sieves, thereby drying theorganic phase comprising the boronic acid and free MIDA ligand, whereinthe method is performed in the deprotection module of an automated smallmolecule synthesizer, said automated small molecule synthesizercomprising: (a) a deprotection module, in fluid communication with (b) adrying and degassing module, in fluid communication with (c) a reactionmodule, in fluid communication with (d) a purification module; (e) atleast one pump which can move liquid from one module to another; and (f)a computer equipped with software, wherein all of the modules are undercontrol of the computer.